3-{2-[2-(3-azaniumylpropoxy)ethoxy]ethoxy}propan-1-aminium di[(2Z)-3-carboxyprop-2-enoate]

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Toxicity to reproduction: oral. 33Oxy.001

Under the conditions of the study, the NOAEL has been determined as 600 mg/kg bw/day in relation to parental systemic toxicity (male/female); 100 mg/kg bw/day in relation to local effect (female – histopathology (lesions in the upper digestive tract at all dose levels) and < 100 mg/kg bw/day in relation to local effect (male – histopathology (lesions in the upper digestive tract at all dose levels).

Toxicity to reproduction: Maleic Acid_RA to Maleic Anhydride.002

Since there was no significant reduction in the percentage of pregnant females or the percentage of fertile males, it is concluded that no adverse effects on fertility were observed with maleic anhydride at doses up to 55 mg/kg/day administered over two generations. Maleic anhydride was toxic to parental animals at all dose levels.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Remarks:

Study has been performed on Bis-Aminopropyl Diglycol aka 3,3'-oxybis(ethyleneoxy)bis(propylamine); (EC 224-207-2; CAS 4246-51-9). This is being used as the read-across substance to Bis Aminopropyl Diglycol Dimaleate; (EC 818-033-1; CAS 1629579-82-3).

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted in accordance with GLP.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

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

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

- 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.

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.

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

Remarks:

Doses / Concentrations:
100, 300 and 1000 (reduced to 600 on study day 7) mg/kg bw/d
Basis: Actual ingested

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.

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:

effects observed, non-treatment-related

Description (incidence and severity):

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All groups:
No rat died prematurely in the present study.
No test substance-related, adverse findings were noted.
Reproductive Performance: No test substance-related, adverse findings were noted.
Clinical Pathology: No test substance-related, adverse findings were noted.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All groups:
No rat died prematurely in the present study.
No test substance-related, adverse findings were noted.
Reproductive Performance: No test substance-related, adverse findings were noted.
Clinical Pathology: No test substance-related, adverse findings were noted.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
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):

FOOD CONSUMPTION AND COMPOUND INTAKE / TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
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 specified

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

effects observed, non-treatment-related

Description (incidence and severity):

WATER CONSUMPTION AND COMPOUND INTAKE
No test substance-related, adverse findings were noted.

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

HAEMATOLOGY / CLINICAL CHEMISTRY / URINALYSIS
See clinical pathology

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

HAEMATOLOGY / CLINICAL CHEMISTRY / URINALYSIS
See clinical pathology

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

OTHER FINDINGS (PARENTAL ANIMALS)
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.

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All groups:
No rat died prematurely in the present study.
No test substance-related, adverse findings were noted.
Reproductive Performance: No test substance-related, adverse findings were noted.
Clinical Pathology: No test substance-related, adverse findings were noted.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
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 / TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
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.


WATER CONSUMPTION AND COMPOUND INTAKE
No test substance-related, adverse findings were noted.


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
--

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
--

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
--

ORGAN WEIGHTS (PARENTAL ANIMALS)
No remarks.


GROSS PATHOLOGY (PARENTAL ANIMALS) / HISTOPATHOLOGY (PARENTAL ANIMALS)
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. 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 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 a local irritating effect but no systemic toxicity.


NEUROBEHAVIOUR
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.


HAEMATOLOGY / CLINICAL CHEMISTRY / URINALYSIS
See clinical pathology


OTHER FINDINGS (PARENTAL ANIMALS)
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: Starting dose: 1000 mg/kg bw/d, reduced to 600 mg/kg bw/d on study day 7

Dose descriptor:

NOAEL

Remarks:

local effect

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Histopathology (lesions in the upper digestive tract at higer dose levels)

Dose descriptor:

NOAEL

Remarks:

local effect

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Histopathology (lesions in the upper digestive tract at all dose levels)

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: 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

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

other: upper digestive tract

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Please see "Details on results (F1)

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Please see "Details on results (F1)

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Please see "Details on results (F1)

Food consumption and compound intake (if feeding study):

not specified

Description (incidence and severity):

Please see "Details on results (F1)

Food efficiency:

not specified

Description (incidence and severity):

Please see "Details on results (F1)

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

not specified

Description (incidence and severity):

Please see "Details on results (F1)

Ophthalmological findings:

not examined

Haematological findings:

not specified

Description (incidence and severity):

Please see "Details on results (F1)

Clinical biochemistry findings:

not specified

Description (incidence and severity):

Please see "Details on results (F1)

Urinalysis findings:

not examined

Sexual maturation:

not specified

Description (incidence and severity):

Please see "Details on results (F1)

Anogenital distance (AGD):

not specified

Description (incidence and severity):

Please see "Details on results (F1)

Nipple retention in male pups:

not specified

Description (incidence and severity):

Please see "Details on results (F1)

Organ weight findings including organ / body weight ratios:

not specified

Description (incidence and severity):

Please see "Details on results (F1)

Gross pathological findings:

not specified

Description (incidence and severity):

Please see "Details on results (F1)

Histopathological findings:

not specified

Description (incidence and severity):

Please see "Details on results (F1)

Other effects:

not specified

Description (incidence and severity):

Please see "Details on results (F1)

Behaviour (functional findings):

not specified

Description (incidence and severity):

Please see "Details on results (F1)

Developmental immunotoxicity:

not examined

VIABILITY (OFFSPRING)
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.

CLINICAL SIGNS (OFFSPRING)
The litter of female animal No. 104 (control group) 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.

BODY WEIGHT (OFFSPRING)
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.

GROSS PATHOLOGY (OFFSPRING)
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.

OTHER FINDINGS (OFFSPRING)
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.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

gross pathology

Key result

Critical effects observed:

not specified

Key result

Reproductive effects observed:

not specified

Conclusions:

Under the conditions of the study, the NOAEL has been determined as 600 mg/kg bw/day in relation to parental systemic toxicity (male/female); 100 mg/kg bw/day in relation to local effect (female – histopathology (lesions in the upper digestive tract at all dose levels) and < 100 mg/kg bw/day in relation to local effect (male – histopathology (lesions in the upper digestive tract at all dose levels).

Executive summary:

An OECD 422 study has been performed on the substance Bis-Aminopropyl Diglycol (also known as 3,3'-oxybis(ethyleneoxy)bis(propylamine); EC Number 224-207-2; CAS Number 4246-51-9). The study is considered as a guideline study conducted in accordance with GLP.

The Bis-Aminopropyl Diglycol was administered to Wistar- strain rats via gavage at concentrations of 100, 300 and 1000 (reduced to 600 on study day 7) mg/kg bw/day. 

Cage-side observations and detailed clinical observations were recorded. Body weight, food consumption, clinical chemistry including haematology and gross pathology were noted to being treatment-related.

Under the conditions of the study, the NOAEL has been determined as 600 mg/kg bw/day in relation to parental systemic toxicity (male/female); 100 mg/kg bw/day in relation to local effect (female – histopathology (lesions in the upper digestive tract at all dose levels) and < 100 mg/kg bw/day in relation to local effect (male – histopathology (lesions in the upper digestive tract at all dose levels).

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Data from handbook or collection of data, read-across from maleic anhydride

Justification for type of information:

Maleic anhydride. However, maleic anhydride hydrolyses under test conditions. As a result, it is believed that maleic acid and its salts were the test materials investigated in this study.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

not specified

GLP compliance:

not specified

Specific details on test material used for the study:

Maleic anhydride (99.7%) Maleic anhydride hydrolyses under test conditions. As a result, it is believed that maleic acid and its salts were the test materials investigated in this study.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

not specified

Duration of treatment / exposure:

Pre-mating and throughout mating, gestation and lactation
Premating exposure period: F0 and F1, a minimum of 80 days

Frequency of treatment:

7 days/week

Remarks:

Doses / Concentrations:
0, 20, 55 and 150 mg/kg
Basis: actual ingested

Control animals:

yes

Details on study design:

dose volume: 10 mL/kg

Parental animals: Observations and examinations:

no data

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

no data

Litter observations:

no data

Postmortem examinations (parental animals):

no data

Postmortem examinations (offspring):

no data

Statistics:

no data

Reproductive indices:

no data

Offspring viability indices:

no data

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

mortalities at 150 mg/kg

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

mortalities at 150 mg/kg

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced at 55 and 150 mg/kg

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

reduced at 55 and 150 mg/kg

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

kidney, bladder

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

reduced at several timepoints, no dose-related pattern

With the exception of a few cases of respiratory rales, the clinical appearance and behavior were not remarkably different from the controls. Significant mortality occurred in adults of both sexes from the 150 mg/kg group.
Body weights in the F0 high-dose group were significantly reduced by Week 11 and this reduction persisted for the remainder of the test. The mid-dose group means were low but not statistically significantly different from the controls.
Macroscopic and microscopic examination of tissues from F0 adults revealed compound-related changes in the kidneys and bladder of rats from all dose groups. However, renal cortical necrosis, present in 60% of the males and in 15% of the females from this group, was observed in the high-dose group only.

Key result

Dose descriptor:

LOEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

respiratory rales

Dermal irritation (if dermal study):

not specified

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

mortality (females) in study week 42

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

most prominent in high dose males

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

absolute kidney weights (females, 20 and 55 mg/kg) increased

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

F1 clinical observations: Respiratory rates were also observed and the incidence and severity appeared to increase with dose. These rats often vigorously resisted handling at the time of dosing. Due to 100% mortality among the high-dose females by study week 42, the remaining high-dose males were terminated by study week 44.
F1 generation showed a similar pattern of depressed weight gain as the F0 generation; however, only the F1 males in the high-dose group had significantly significant body weight depression at 30 weeks. No fertility or offspring data are available from the high-dose animals.
In the F1 generation, the absolute kidney weights of adult females in the low- and mid-dose groups were significantly increased to 108 to 111%, respectively, of the control value. There were no microscopic changes in these kidneys.
Fertility was significantly reduced in the treatment groups at several timepoints; however, neither a dose-related reduction nor a pattern within a generation suggested a treatment-related effect
No adverse effects on litter size and on pup survival were observed at doses up to 150 mg/kg/day in the F1 litters, or at 55 mg/kg/day in the F2 litters. Microscopic examination of tissues from pups in the F2 liters revealed no treatment-related changes. Therefore, treatment up to 55 mg/kg/day for two generations had no adverse effect on pups.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: litter size, pup survival

Key result

Dose descriptor:

NOEL

Generation:

F2

Effect level:

55 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: litter size, pup survival

Key result

Reproductive effects observed:

not specified

Conclusions:

Since there was no significant reduction in the percentage of pregnant females or the percentage of fertile males, it is concluded that no adverse effects on fertility were observed with maleic anhydride at doses up to 55 mg/kg/day administered over two generations. Maleic anhydride was toxic to parental animals at all dose levels.

Executive summary:

A two-generation study similar to OECD 416 was performed with maleic anhydride in Sprague-Dawley rats. The test substance was administered orally at doses of 0, 20, 55 and 150 mg/kg body weight and day, on 7 days/week, at a dose volume of 10 mL/kg body weight.

Results:

F0 clinical observations: with the exception of a few cases of respiratory rales, the clinical appearance and behavior were not remarkably different from the controls. Significant mortality occurred in adults of both sexes from the 150 mg/kg group.

F1 clinical observations: Respiratory rates were also observed and the incidence and severity appeared to increase with dose. These rats often vigorously resisted handling at the time of dosing.  Due to 100% mortality among the high-dose females by study week 42, the remaining high-dose males were terminated by study week 44.

Body weights in the F0 high-dose group were significantly reduced by Week 11 and this reduction persisted for the remainder of the test. The mid-dose group means were low but not statistically significantly different from the controls. F1 generation showed a similar pattern of depressed weight gain; however, only the F1 males in the high-dose group had significantly significant body weight depression at 30 weeks. No fertility or offspring data are available from the high-dose animals.

Macroscopic and microscopic examination of tissues from F0 adults revealed compound-related changes in the kidneys and bladder of rats from all dose groups. However, renal cortical necrosis, present in 60% of the males and in 15% of the females from this group, was observed in the high-dose group only. In the F1 generation, the absolute kidney weights of adult females in the low- and mid-dose groups were significantly increased to 108 to 111%, respectively, of the control value. There were no microscopic changes in these kidneys.

Fertility was significantly reduced in the treatment groups at several timepoints; however, neither a dose-related reduction nor a pattern within a generation suggested a treatment-related effect.

No adverse effects on litter size and on pup survival were observed at doses up to 150 mg/kg/day in the F1 litters, or at 55 mg/kg/day in the F2 litters. Microscopic examination of tissues from pups in the F2 liters revealed no treatment-related changes. Therefore, treatment up to 55 mg/kg/day for two generations had no adverse effect on pups.

Since there was no significant reduction in the percentage of pregnant females or the percentage of fertile males, it is concluded that no adverse effects on fertility were observed with maleic anhydride at doses up to 55 mg/kg/day administered over two generations. Maleic anhydride was toxic to parental animals at all dose levels.

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

other information

Justification for type of information:

The read-across prediction was based upon the data available on the source substances. The two source substances Maleic Acid and 3,3'-oxybis(ethyleneoxy)bis(propylamine 4,7,10-Trioxatridecan-1,13-diam are mixed together in water in a 2:1 ratio to form the target substance. The only differences are the charged species and that the target substance only exists in an aqueous solution. The source and target substances are all mono-constituent substances. The remaining source substance, Maleic Anhydride was used because this is the substance that can be found in the disseminated ECHA dossier for Maleic Acid, and that Maleic Anhydride hydrolyses under test conditions. As a result, it is believed that Maleic Acid and its salts were the test materials investigated in the studies.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Read across values from the most suitable substances have been used for classification.

The substance does not meet the criteria for classification for toxicity to reproduction under CLP.