3,3'-oxybis(ethyleneoxy)bis(propylamine)

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:

Jan - Dec 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

toxicity to reproduction

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan - Dec 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

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: EPA, Health Effects Test Guidelines; 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)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht; 22.10.2009

Limit test:

no

Specific details on test material used for the study:

- Name of test substance: 3,3'-Oxybis(ethyleneoxy)bis(propylamine)
- Test substance No.: 11/0401-1
- Batch identification: O 2895
- Purity: 99.7% (project No: 11L00203)
- Homogeneity: given (visually, project No: 11L00203)
- Stability: expiry date: 15 Jun 2013
- Date of production: 15 Jun 2011
- Physical state/ appearance: liquid / colorless, clear
- Storage conditions: room temperature
- Other: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is the preferred animal species for reproduction studies according to the various test guidelines and the Wistar strain was selected.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at supplied: 10-11 weeks
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Weight at study initiation: Males: 321 - 322 g, Females 205 - 209 g
- Fasting period before study: no
- Housing: individually in Makrolon type M III cages;
- Exceptions:
- During overnight matings, male and female mating partners were housed together in Makrolon type M III cages
- Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- For motor activity (MA) measurements the animals were housed individually in polycarbonate cages (floor area of about 800 cm2) and
small amounts of bedding material
- The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet, ad libitum: Ground Kliba maintenance diet mouse-rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water, ad libitum: drinking water
- Acclimation period: On the day of arrival the animals were subjected to an appropriate acclimatization period.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 30-70%
- Air changes (per hr): 15
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 2012-01-09 To: 2013-03-12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a magnetic stirrer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least once a week and were stored at room temperature.

VEHICLE
- Justification for use and choice of vehicle: solubility
- Concentration in vehicle:
1.00 mg/100 mL (100 mg/kg bw/d)*, 3.00 mg/100 mL (300 mg/kg bw/d)*, 10.00 and 6.00 mg/100 mL (1000 and 600 mg/kg bw/d)*
*) The dose refers to the body weight of the individual rats determined most recently.
- Administration volume:: 10 mL/kg

Details on mating procedure:

F0 generation parental animals and their progeny
The animals of the control group were treated in the same way with the vehicle only (drinking water). The calculation of the administered volume was generally based on the most recent individual body weights. Fourteen days after the beginning of treatment, males and females from the same test group were mated overnight in a ratio of 1:1 (details of pairing see 3.7.2.). On study day 49, a functional observational battery and motor activity measurement were carried out in the first five surviving parental male animals per group. The females were allowed to litter and rear their pups until day 4 after parturition. On PND 4, all pups were sacrificed and examined. On study day 56, a functional observational battery and motor activity measurement was carried out in the first five surviving parental female animals (with litter) per group. From the first 5 surviving parental male animals per group and the first 5 surviving parental female animals which delivered first urinalysis was carried out on study days 51 (males) and 58 (females). Clinicochemical and hematological examinations were carried out on study days 60 (males) and 63 (females). At the end of the study (males: study day 60, females: study day 63), the animals were sacrificed after a fasting period (withdrawal of food) for at least 16-20 hours.

Mating of F0 generation parental animals
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group.
The animals were paired by placing the female in the cage of the male mating partner from about 15.00 h until 07.00-09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The stability of the test substance in drinking water for a period of 7 days at room temperature was proven during the study (BASF project No. 01Y0401/11Y018; see PART III, Supplement). Homogeneity and concentration control analyses of the test-substance preparations were performed in all concentrations at the start of the administration period. Additionally, samples from all concentrations as reverse samples for concentration control analysis were taken at the end of the study.
The determined mean and single concentrations of the test substance in drinking water were found to be in the range of 90% – 110% of the nominal
concentrations. These results demonstrated the correctness of the concentrations.

Duration of treatment / exposure:

After the acclimatization period, the test substance was administered orally via gavage to the F0 generation parental animals, daily at the same time in the morning (exception: no administration to animals being in labor). The treatment lasted up to one day prior to sacrifice. The animals of the control group were treated in the same way with the vehicle only (drinking water).

Frequency of treatment:

Daily

Details on study schedule:

Age at mating of the mated animals in the study: 13-14 weeks

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

analytical verified concentration

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

analytical verified concentration

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

reduced to 600 mg/kg bw/day on study day 7; analytical verified concentration

No. of animals per sex per dose:

10 animals

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Testing up to the limit dose of 1000 mg/kg bw (dose reduction due to local toxicity of the test item)
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
- Fasting period before blood sampling for clinical biochemistry: 16 hours

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal.
The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.
On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.
The day of littering was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high).
The following parameters were examined: abnormal behavior when handled, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
- During the mating period the parental fe¬males were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
- Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly. These body weight data were
solely used for the calculations of the dose volume.


FOOD CONSUMPTION AND COMPOUND INTAKE:
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period (male and female F0 animals)
- Food consumption of the F0 females with evidence of sperm was determined on GD 0-7, 7-14, 14-20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 1-4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In the morning blood was taken from the retroorbital venous plexus from fasted animals.
The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany). The blood sampling procedure and
subsequent analysis of blood and serum samples were carried out in a randomized sequence.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Parameters were determined in blood with EDTA K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany)
- Parameters examined: see Table 1 in 'Any other information on materials and methods incl. tables'.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In the morning blood was taken from the retroorbital venous plexus from fasted animals.
- Animals fasted: Yes
- How many animals: first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters
- Parameters examined: see Table 2 in 'Any other information on materials and methods incl. tables'.

URINALYSIS: Yes
- Time schedule for collection of urine: overnight
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- The dry chemical reactions on test strips (Combur 10 test M, Roche, Mannheim, Germany) used to determine urine constituents semiquantitatively
were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany).
- Parameters examined: see Table 3 in 'Any other information on materials and methods incl. tables'.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Functional observational battery (FOB) was performed in all animals towards the end of the administration period
- Dose groups that were examined: all animals

- Battery of functions tested:
Home cage observations:
Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Gait abnormalities.
Open field observations:
Behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, feces (number of fecal pellets/appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes, number of rearings within two minutes.
Sensorimotor tests/reflexes:
Approach response, touch response, vision ("visual placing response"), pupillary reflex, pinna reflex, audition ("startle response"), coordination of movements ("righting response"), behavior during "handling", vocalization, pain perception ("tail pinch"), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings.

Motor activity (MA) measured on the same day as the FOB was performed.

Oestrous cyclicity (parental animals):

No data.

Sperm parameters (parental animals):

Parameters examined in male parental generation:
- testis weight
- epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no; pups were scheduled sacrifice on PND 4

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups
- stillbirths
- live births
- postnatal mortality
- presence of gross anomalies
- weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS: yes
All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2.
All pups were examined externally and eviscerated; their organs were assessed macroscopically.
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation.
Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
The following parameters were examined in F1 offspring:
- number and sex of pups
- stillbirths
- live births
- postnatal mortality
- presence of gross anomalies
- weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS: yes
- for external and internal abnormalities
- possible cause of death wast determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE /GROSS NECROPSY
All surviving parental animals (males/females) were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Gross necropsy consisted of external and internal examinations.


HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights: Weight assessment was carried out on all animals.
- The following weights were determined: Anesthetized animals, Epididymides, Testes.
- The following weights were determined in 5 animals/sex and test group (females with litters, same animals as used for clinical pathology examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus
The following organs/ tissues were preserved in neutral-buffered 4% formaldehyde or in modified Davidson’s solution: Adrenal glands, All gross lesions, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Eyes with optic nerve, Esophagus, Extraorbital lacrimal gland, Epididymides (modified Davidson’s solution), Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands, (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Target organs,Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina.

For further evaluation proceeding, see Table 4 in 'Any other information on materials and methods incl. tables'.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was scheduled sacrifice on PND 4 under isoflurane anesthesia with CO2.
- These animals were subjected to postmortem examinations as follows: All pups were examined externally and eviscerated; their organs were assessed macroscopically. All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

GROSS NECROPSY
- Gross necropsy consisted of external examination

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed

Statistics:

Please refer to 'Any other information on materials and methods incl. tables'.

Reproductive indices:

Male reproduction data
The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs. For the males, mating and fertility indices were calculated for F1 litters.

Male mating index (%) = (number of males with confirmed mating* / number of males placed with females) x 100
*defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = (number of males proving their fertility* / number of males placed with females x 100
*defined by a female with implants in utero


Female reproduction and delivery data
The pairing partners, the number of mating days until vaginal sperm were detected, and gestational status were recorded for F0 females.
For the females, mating, fertility and gestation indices were calculated for F1 litters.

Female mating index (%) = (number of females mated* / number of females placed with males) x 100
*defined as the number of females with vaginal sperm or with implants in utero

Female fertility index (%) = (number of females pregnant* / number of females mated**) x 100
*defined as the number of females with implants in utero
**defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant*) x 100
*defined as the number of females with implants in utero


The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters.

Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100

Offspring viability indices:

Pup viability/mortality: The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1 - 4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4.

Viability index (%) = (number of live pups on day 4 after birth ./. number of live pups on the day of birth) x 100


Sex ratio: On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at day 0 and day 4 after birth.

Sex ratio = (number of live male or female pups on day 0/4 ./. number of live male and female pups on day 0/4) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related, adverse findings were noted.
A temporary, short appearance of salivation immediately after dosing was concluded to be induced by a bad taste of the test substance or local affection of the upper digestive tract.

Mortality:

no mortality observed

Description (incidence):

No rat died prematurely in the present study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

On study day 7 (premating) mean body weight of male animals in test group 3 (1000 and 600 mg/kg bw/d) was significantly decreased (-6%). Although not significantly altered, mean body weight loss in female animals was observed after 7 days of treatment.
Mean body weight change values during premating were significantly decreased in male animals of test group (1000 and 600 mg/kg bw/d) between study days 0-7 (-84%) and 0-13 (-48%). Although not significantly altered mean body weight change value in female animals was observed between study days 0-7 days. All mentioned findings were assessed as being related to treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In test group 3 (1000 and 600 mg/kg bw/d) food consumption during premating was significantly decreased in male animals between study days 0-7 (-17%) and 0-13 (-11%) as well as in female animals between study days 0-7 (-19%). These findings were assessed as being related to treatment with the test substance by irritating the upper digestive tract.
No other findings were observed for male and female animals in test group 1 and 2 (100 and 300 mg/kg bw/d) nor for male and female animals of test group 3 (1000 and 600 mg/kg bw/d) after reducing the dose level.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Home cage observations: No test substance-related effects were observed.
Open field observations: No test substance-related effects were observed.
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative Parameters: No test substance-related effects were observed.
Motor activity measurement: There were no significant deviations concerning the overall motor activity (summation of all intervals) and regarding the single intervals in male and female animals of all test groups in comparison to the concurrent control group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment of male and female Wistar rats with up to 1000 and 600 mg/kg bw/d of the test substance led to treatment-related findings in the upper digestive tract (starting at 100 mg/kg bw/d in males and 300 mg/kg bw/d in females). The hyperplasia, hydropic degeneration of squamous cells and inflammatory cell infiltrates, occasionally with multinucleated giant cells in the forestomach as well as the hyperemia, eosinophilic cytoplasmic change, increased mitotic figures, submucosal edema and inflammatory cell infiltrates in the glandular stomach were regarded to be signs of a slight irritating effect of the test substance. These findings were almost exclusively observed around or at the margo plicatus and in the glandular stomach in the fundic area. They were regarded to be adverse. The increase in thickness in the duodenum (only males >= 300 mg/kg bw/d) was also regarded to be treatment-related and as the specific mechanism could not be determined, it was also judged to be adverse in nature. All these adverse findings described above are regarded to be local irritating effects but no systemic toxicity.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

- testis weight and epididymis weight were unaffected by treatment

Reproductive performance:

no effects observed

Description (incidence and severity):

The male fertility index ranged between 80% and 100% (reflects the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data)
The female mating index calculated after the mating period for F1 litter was 100% for all test groups (0, 100, 300, 1000 and 600 mg/kg bw/d). The mean duration until sperm was
detected (GD 0) was 3.8, 3.0, 2.9 and 2.8 days in test groups 0-3 (0, 100, 300, 1000 and 600mg/kg bw/d, respectively).
The female fertility index varied between 80% and 100%.
The mean duration of gestation was between 22.0 and 22.3 days and did not show significant differences.
The gestation index reached 100% in all test groups including the control group.
The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 99% (control group) and 100% (test groups 1, 2 and 3). Single stillborn pups were
only seen in control group.

No findings on the reproduction tract were observed. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Dose descriptor:

NOAEL

Remarks:

parental systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect up to and including the highest tested dose (starting dose: 1000 mg/kg bw/d, reduced to 600 mg/kg bw/d on study day 7)

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect up to and including the highest tested dose (starting dose: 1000 mg/kg bw/d, reduced to 600 mg/kg bw/d on study day 7)

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Starting dose: 1000 mg/kg bw/d, reduced to 600 mg/kg bw/d on study day 7

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The litter of one control female showed reduced nutritional condition. One pup of female animal No. 123 (test group 2, 300 mg/kg bw/d) showed skin lesion on right flank and hindlimb. Two pups of control group and one pup of test group 2 (300 mg/kg bw/d) were not assessed because they died during interval. The surviving F1 pups of any test group did not show clinical signs up to scheduled sacrifice on PND 4.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The viability index as indicator for pup mortality between PND 0 and 4 was 100% for test groups 1 and 3 (100 as well as 1000 and 600 mg/kg bw/d). In control group the viability index was 99% because of 1 pup found dead. In test group 2 (300 mg/kg bw/d) the viability index was 97% because of 2 pups found dead and 2 cannibalized pup in 3 different litters. The remaining pups which belonged to this animal did not show any findings until sacrifice.
The viability index was still within the historical control data and reflected the normal range of biological variation inherent in the strain used in this study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the concurrent control values.Two female and 10 male runts were seen in one litter in control group on PND1. One female runt were seen in test groups 2 and 3 (300 as well as 1000 and 600 mg/kg bw/d) on PND 1. All values were within the range of the biological variation inherent in the strain of rats used for this study.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Post mortem autolysis was observed for several pups in all test groups including the control. In 2 pups of test group 2 (300 mg/kg bw/d; female Nos. 127 and 130) a discolored liver lobe was observed. This finding was assessed as being spontaneous in nature and without biological relevance. Two pups of test group 2 (300 mg/kg bw/d; female Nos. 128 and 130) were not assessed because they were cannibalized on PND 0.

Histopathological findings:

not examined

Description (incidence and severity):

The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between test groups

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

The mean number of delivered pups per dam and the rate of liveborn and stillborn pups reflect the normal range of biological variation inherent in the strain used in this study. The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between test groups

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental toxicity up to and including the highest tested dose

Reproductive effects observed:

not specified

Conclusions:

The no observed adverse effect level (NOAEL) for general systemic toxicity was 600 mg/kg bw/d in males and in females, the NOAEL for the locally irritating effect in the stomach was 100 mg/kg bw/d in females and below that in males. The NOAEL for reproductive performance and fertility was set to 600 mg/kg bw/d in male and
female Wistar rats. The NOAEL for developmental toxicity was effective 600 mg/kg bw/d.

Executive summary:

In this reliabe OECD 422 study performed in compliance with GLP, the test substance was administered orally via gavage to groups of 10

male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). Because of body weight loss in several animals of both sexes in test group 3 (1000 mg/kg bw/d) the animals were treated with 600 mg/kg bw/d from study day 7 onwards. Drinking water served as vehicle. The objective of the study was to detect possible effects of the test substance on the integrity and performance of male and female reproductive systems including gonadal function, mating behavior, conception, gestation and parturition. Furthermore, it was intended to obtain information about the general toxicological profile including target organs and the no observed adverse effect level (NOAEL) after repeated oral administration. The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 4 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice.

Parental animals of the highest dose (1000 mg(kg bw(d) showed piloerection after treatment (1 of 10 male animal and 2 of 10 female animals) during the first study week. Food consumption during premating was significantly decreased in male animals between study days 0-7 (-17%) and 0-13 (-11%) as well as in female animals between study days 0-7 (-19%). Mean body weight of male animals was significantly decreased (-6%) on study day 7. Although not significantly altered mean body weight loss in female animals was observed after 7 days of treatment. Mean body weight change values during premating were significantly decreased in male animals between study days 0-7 (-84%) and 0-13 (-48%). Although not significantly reduced mean body weight change value in female animals was observed between study days 0-7 days. After exposure dose reduction from study day 7 (600 mg/kg bw/d) onwards, solely piloerection was observed in 1 of 10 male animals during the second study week. In the lower dose groups (100 and 300 mg/kg bw/d) no substance-related adverse findings in respect of clinical examinations and clinical pathology were observed. Local toxicity was observed after substance administration for all exposure settings. Target organ was the stomach for male and female animals and, in addition, the duodenum for males. The hyperplasia, hydropic degeneration of squamous cells and inflammatory cell infiltrates, occasionally with multinucleated giant cells in the forestomach as well as the hyperemia, eosinophilic cytoplasmic change, increased mitotic figures, submucosal edema and inflammatory cell infiltrates in the glandular stomach were regarded to be signs of a slight irritating effect of the test substance. These findings were almost exclusively observed around or at the margo plicatus and in the glandular stomach in the fundic area. They were regarded to be adverse. The increase in thickness in the duodenum (only males >= 300 mg/kg bw/d) was also regarded to be treatment-related and as the specific mechanism could not be determined, it was also judged to be adverse in nature. All these adverse findings described above are regarded to be local irritating effects but no systemic toxicity.

The reproductive performance of the parental animals was unaffected by the treatment. The male fertility index ranged between 80% and 100%. The female mating index was 100% for all test groups. The female fertility index varied between 80% and 100%. The mean duration of gestation was between 22.0 and 22.3 days and did not show significant differences. In all test groups the gestation index reached 100% in all test groups including the control group.

The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 99% (control group) and 100% (test groups 1, 2 and 3). Single stillborn pups were only seen in control group.

Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 600 mg/kg bw/d in males and in females, the NOAEL for the locally irritating effect in the stomach was 100 mg/kg bw/d in females and below that in males. The NOAEL for reproductive performance and fertility was set to 600 mg/kg bw/d in male and female Wistar rats. The NOAEL for developmental toxicity was effective 600 mg/kg bw/d.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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: EPA, Health Effects Test Guidelines; 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)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht; 22.10.2009

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,3'-oxybis(ethyleneoxy)bis(propylamine)

EC Number:

224-207-2

EC Name:

3,3'-oxybis(ethyleneoxy)bis(propylamine)

Cas Number:

4246-51-9

Molecular formula:

C10H24N2O3

IUPAC Name:

3,3'-[oxybis(ethane-2,1-diyloxy)]dipropan-1-amine

Specific details on test material used for the study:

- Name of test substance: 3,3'-Oxybis(ethyleneoxy)bis(propylamine)
- Test substance No.: 11/0401-1
- Batch identification: O 2895
- Purity: 99.7% (project No: 11L00203)
- Homogeneity: given (visually, project No: 11L00203)
- Stability: expiry date: 15 Jun 2013
- Date of production: 15 Jun 2011
- Physical state/ appearance: liquid / colorless, clear
- Storage conditions: room temperature
- Other: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is the preferred animal species for reproduction studies according to the various test guidelines and the Wistar strain was selected.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at supplied: 10-11 weeks
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Weight at study initiation: Males: 321 - 322 g, Females 205 - 209 g
- Fasting period before study: no
- Housing: individually in Makrolon type M III cages;
- Exceptions:
- During overnight matings, male and female mating partners were housed together in Makrolon type M III cages
- Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- For motor activity (MA) measurements the animals were housed individually in polycarbonate cages (floor area of about 800 cm2) and
small amounts of bedding material
- The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet, ad libitum: Ground Kliba maintenance diet mouse-rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water, ad libitum: drinking water
- Acclimation period: On the day of arrival the animals were subjected to an appropriate acclimatization period.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 30-70%
- Air changes (per hr): 15
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 2012-01-09 To: 2013-03-12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a magnetic stirrer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least once a week and were stored at room temperature.

VEHICLE
- Justification for use and choice of vehicle: solubility
- Concentration in vehicle: 1.00 mg/100 mL (100 mg/kg bw/d)*, 3.00 mg/100 mL (300 mg/kg bw/d)*, 10.00 and 6.00 mg/100 mL (1000 and 600 mg/kg bw/d)*
*) The dose refers to the body weight of the individual rats determined most recently.
- Administration volume:: 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in drinking water for a period of 7 days at room temperature was proven during the study. Homogeneity and concentration control analyses of the test-substance preparations were performed in all concentrations at the start of the administration period. Additionally, samples from all concentrations as reverse samples for concentration control analysis were taken at the end of the study. The various analyses confirmed the stability over 7 days and homogeneity and concentration in the vehicle.
The determined mean and single concentrations of the test substance in drinking water were found to be in the range of 90 % – 110 % of the nominal
concentrations. These results demonstrated the correctness of the concentrations.

Duration of treatment / exposure:

Males were exposed for 59 days (prior to mating, during mating, and up to termination) and females 62 days (prior to mating, during mating, during post-coitum, and at least 4 days of lactation).

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

analytical verified concentration

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

analytical verified concentration

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

reduced to 600 mg/kg bw/day on study day 7; analytical verified concentration

No. of animals per sex per dose:

10 animals

Control animals:

yes, concurrent vehicle

Details on study design:

The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
Fasting period before blood sampling for clinical biochemistry: 16 hours

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity.
The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams.
On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high).
The following parameters were examined: abnormal behavior when handled, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
- Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION:
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period (male and female F0 animals)
- Food consumption of the F0 females with evidence of sperm was determined on GD 0-7, 7-14, 14-20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 1-4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In the morning towards the end of the administrative period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Parameters examined: see Table 1 in 'Any other information on materials and methods incl. tables'.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In the morning towards the end of the administrative period
- Animals fasted: Yes
- How many animals: first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Parameters examined: see Table 2 in 'Any other information on materials and methods incl. tables'.

URINALYSIS: Yes
- Time schedule for collection of urine: overnight towards the end of the administrative period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: see Table 3 in 'Any other information on materials and methods incl. tables'.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Functional observational battery (FOB) was performed in all animals towards the end of the administration period
- Dose groups that were examined: all animals

- Battery of functions tested:
Home cage observations:
Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Gait abnormalities.
Open field observations:
Behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, feces (number of fecal pellets/appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes, number of rearings within two minutes.
Sensorimotor tests/reflexes:
Approach response, touch response, vision ("visual placing response"), pupillary reflex, pinna reflex, audition ("startle response"), coordination of movements ("righting response"), behavior during "handling", vocalization, pain perception ("tail pinch"), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings.

Motor activity (MA) measured on the same day as the FOB was performed.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
Organ weights: Weight assessment was carried out on all animals.
- The following weights were determined: Anesthetized animals, Epididymides, Testes.
- The following weights were determined in 5 animals/sex and test group (females with litters, same animals as used for clinical pathology examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus

HISTOPATHOLOGY: Yes
The following organs/ tissues were preserved in neutral-buffered 4% formaldehyde or in modified Davidson’s solution: Adrenal glands, All gross lesions, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Eyes with optic nerve, Esophagus, Extraorbital lacrimal gland, Epididymides (modified Davidson’s solution), Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands, (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Target organs,Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina.
For further evaluation proceeding, see Table 4 in 'Any other information on materials and methods incl. tables'.

Other examinations:

The supplier assayed the food used in the study for chemical and microbiological contaminants.
The drinking water is regularly assayed for chemical contaminants.
The bedding and the enrichment are regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).

Statistics:

Please refer to 'Any other information on materials and methods incl. tables'.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related, adverse findings were noted.
A temporary, short appearance of salivation immediately after dosing was concluded to be induced by a bad taste of the test substance or local affection of the upper digestive tract.

Mortality:

no mortality observed

Description (incidence):

No rat died prematurely in the present study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

On study day 7 (premating) mean body weight of male animals in test group 3 (1000 and 600 mg/kg bw/d) was significantly decreased (-6%). Although not significantly altered, mean body weight loss in female animals was observed after 7 days of treatment.
Mean body weight change values during premating were significantly decreased in male animals of test group (1000 and 600 mg/kg bw/d) between study days 0-7 (-84%) and 0-13 (-48%). Although not significantly altered mean body weight change value in female animals was observed between study days 0-7 days. All mentioned findings were assessed as being related to treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In test group 3 (1000 and 600 mg/kg bw/d) food consumption during premating was significantly decreased in male animals between study days 0-7 (-17%) and 0-13 (-11%) as well as in female animals between study days 0-7 (-19%). These findings were assessed as being related to treatment with the test substance by irritating the upper digestive tract.
No other findings were observed for male and female animals in test group 1 and 2 (100 and 300 mg/kg bw/d) nor for male and female animals of test group 3 (1000 and 600 mg/kg bw/d) after reducing the dose level.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Home cage observations: No test substance-related effects were observed.
Open field observations: No test substance-related effects were observed.
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative Parameters: No test substance-related effects were observed.
Motor activity measurement: There were no significant deviations concerning the overall motor activity (summation of all intervals) and regarding the single intervals in male and female animals of all test groups in comparison to the concurrent control group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The mean absolute weights of the kidney were significantly increased in females of the highest dose (9% increased compared to control). The increase in absolute kidney weights of females of test group 3 (1000 and 600 mg/kg bw/d) was not regarded to be treatment-related due to a missing histopathological correlate and a missing dose-response relationship.
The increase of relative liver weights in females of test group 2 (300 mg/kg bw/d) by 6% was also not regarded to be treatment-related because the livers examined histopathologically in
animals of test group 3 (1000 and 600 mg/kg bw/d) did not show any finding which could explain the weight increase.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Glandular stomach (discoloration, thickening of wall), duodenum (thickening of wall)
The obove mentioned gross lesions in males in the glandular stomach and duodenum were regarded to be treatment related. All other gross lesions noted were single observations and they were regarded to have developed spontaneously and unrelated to compound and treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment of male and female Wistar rats with up to 1000 and 600 mg/kg bw/d of the test substance led to treatment-related findings in the upper digestive tract (starting at 100 mg/kg bw/d in males and 300 mg/kg bw/d in females). The hyperplasia, hydropic degeneration of squamous cells and inflammatory cell infiltrates, occasionally with multinucleated giant cells in the forestomach as well as the hyperemia, eosinophilic cytoplasmic change, increased mitotic figures, submucosal edema and inflammatory cell infiltrates in the glandular stomach were regarded to be signs of a slight irritating effect of the test substance. These findings were almost exclusively observed around or at the margo plicatus and in the glandular stomach in the fundic area. They were regarded to be adverse. The increase in thickness in the duodenum (only males >= 300 mg/kg bw/d) was also regarded to be treatment-related and as the specific mechanism could not be determined, it was also judged to be adverse in nature. All these adverse findings described above are regarded to be local irritating effects but no systemic toxicity.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Findings which occurred either individually or were biologically equally distributed over control and treatment groups, were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

parental systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect up to and including the highest tested dose (starting dose: 1000 mg/kg bw/d, reduced to 600 mg/kg bw/d on study day 7)

2

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

3

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

4

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect up to and including the highest tested dose (starting dose: 1000 mg/kg bw/d, reduced to 600 mg/kg bw/d on study day 7)

5

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Starting dose: 1000 mg/kg bw/d, reduced to 600 mg/kg bw/d on study day 7

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 5: Histopathology findings (10 animals in each dose group)

	Males				Females
Test group (mg/kg bw/d)	0	100	300	1000 (600)	0		100	300	1000 (600)
Forestomach
• Hyperplasia squamous, focal			2	4				1
• Degeneration, hydropic, (m)focal		1	2	2		1		3	5
•Inflammatory cell infiltrates, (m)focal			1	6					6
Glandular stomach
• Hyperemia, mucosal		1	1	2
• Eosinophilic cytoplasmic change		1	5	4				1	8
• Mitotic figures increased			4	3				1	3
• Edema, submucosal		2	3	2				1	2
• Inflammatory cell infiltrates, (m)focal		3	5	7				1	6
Duodenum
•Thickness, increased			1	5

Applicant's summary and conclusion

Conclusions:

The no observed adverse effect level (NOAEL) for general systemic toxicity was 600 mg/kg bw/d in males and in females, the NOAEL for the locally
irritating effect in the stomach was 100 mg/kg bw/d in females and below that in males.

Executive summary:

In this reliabe OECD 422 study performed in compliance with GLP (reliability 1), the test substance was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). Because of body weight loss in several animals of both sexes in test group 3 (1000 mg/kg bw/d) the animals were treated with 600 mg/kg bw/d from study day 7 onwards. Drinking water served as vehicle. The objective of the study was to detect possible effects of the test substance on the integrity and performance of male and female reproductive systems including gonadal function, mating behavior, conception, gestation and parturition. Furthermore, it was intended to obtain information about the general toxicological profile including target organs and the no observed adverse effect level (NOAEL) after repeated oral administration. The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 4 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice.

Parental animals of the highest dose (1000 mg(kg bw(d) showed piloerection after treatment (1 of 10 male animal and 2 of 10 female animals) during the first study week. Food consumption during premating was significantly decreased in male animals between study days 0-7 (-17%) and 0-13 (-11%) as well as in female animals between study days 0-7 (-19%). Mean body weight of male animals was significantly decreased (-6%) on study day 7. Although not significantly altered mean body weight loss in female animals was observed after 7 days of treatment. Mean body weight change values during premating were significantly decreased in male animals between study days 0-7 (-84%) and 0-13 (-48%). Although not significantly reduced mean body weight change value in female animals was observed between study days 0-7 days. After exposure dose reduction from study day 7 (600 mg/kg bw/d) onwards, solely piloerection was observed in 1 of 10 male animals during the second study week. In the lower dose groups (100 and 300 mg/kg bw/d) no substance-related adverse findings in respect of clinical examinations and clinical pathology were observed. Local toxicity was observed after substance administration for all exposure settings. Target organ was the stomach for male and female animals and, in addition, the duodenum for males. The hyperplasia, hydropic degeneration of squamous cells and inflammatory cell infiltrates, occasionally with multinucleated giant cells in the forestomach as well as the hyperemia, eosinophilic cytoplasmic change, increased mitotic figures, submucosal edema and inflammatory cell infiltrates in the glandular stomach were regarded to be signs of a slight irritating effect of the test substance. These findings were almost exclusively observed around or at the margo plicatus and in the glandular stomach in the fundic area. They were regarded to be adverse. The increase in thickness in the duodenum (only males >= 300 mg/kg bw/d) was also regarded to be treatment-related and as the specific mechanism could not be determined, it was also judged to be adverse in nature. All these adverse findings described above are regarded to be local irritating effects but no systemic toxicity.

Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 600 mg/kg bw/d in males and in females, the NOAEL for the locally

irritating effect in the stomach was 100 mg/kg bw/d in females and below that in males.