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Description of key information

Repeated dose toxicity:
- oral, screening (OECD422), rat: NOAEL(systemic toxicity) = 75 mg/kg bw (highest dose tested)
- Waiver Rep. dose, 90 d: A screening test (OECD 422) where no effects were observed is available.

No effects were observed at the highest dose tested. Since no further information is to be expected, the substance has corrosive properties, and with regard to animal welfare (reduction of animal testing), the conduct of a sub-chronic (90 days) study is deemed to be not necessary.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
other: dose-range finder
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 293 to 336 g, Females: 205 to 250 g
- Housing: In groups of five or six in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding with paper enrichment. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2018C rodent maintenance diet (Provimi Kliba SA, Kaiseraugst/Switzerland) was available ad libitum.
- Water: Community tap-water from Fuellinsdorf was available ad libitum in water bottles.
- Acclimation period: 7 days

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

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared weekly. Di-(2-ethylhexyl)amine was weighed into a glass beaker on a tared precision balance and the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group (middle only) as well as three samples (top, middle and bottom) of about 0.5 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 0.5 g of each concentration were taken from the middle to confirm the stability. On 16-Aug-2012, samples were taken from the middle to confirm concentration. The samples were analyzed by GC coupled to an FID detector following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Duplicates were taken of all samples. The results indicate the accurate use of the test item di-(2-ethylhexyl)amine and corn oil as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.
Duration of treatment / exposure:
- Males: minimum 4 weeks;
- Females: approximately 6 weeks;
Males were sacrificed after treatment of 28 days, when no longer needed for the assessment of reproductive effects. Pups were sacrificed on day 4 post partum. Dams were sacrificed on day 5 post partum. Since in several females a birth did not occur on the expected date (day 21 post coitum), these dams were sacrificed on day 25 post coitum.
Frequency of treatment:
once daily
Dose / conc.:
7.5 mg/kg bw/day (nominal)
Remarks:
nominal conc. in vehicle
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
No. of animals per sex per dose:
11
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a 14-day dose range-finding study (D52701, BASF project number 01R0454/11X312), four rats per group were exposed by gavage to 30, 100, or 300mg/kg b.w. bis(2-ethylhexyl)amine. All high dose animals died or were euthanized by day 10. These animals showed body weight loss, diarrhea, and signs of pain and extreme discomfort, e.g., salivation, bedding in mouth, hunched posture, and vocalization, which were all attributed to the caustic properties of test substance. Animals receiving 100mg/kg still showed salivation and bedding in mouth. Since the mid dose also gained significantly less weight than the controls, this dose was deemed too high for the longer study duration of an OECD 422 and the use of pregnant females.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Viability / Mortality: twice daily; Clinical signs: once daily, during acclimatization and up to day of necropsy; additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing;
- Cage side observations: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern); changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior;

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration of the test item (day 6 of acclimatization) and weekly thereafter (in the gestation period on day 0, 6, 13 and 20 post coitum), detailed clinical observations were performed outside the home cage in a standard arena;

BODY WEIGHT: Yes
- Time schedule for examinations: daily (from treatment start to day of necropsy)

FOOD CONSUMPTION AND COMPOUND INTAKE: Males: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; after pairing period days 1 - 8 and 8 - 11; Females: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; gestation days 0 – 7, 7 - 14 and 14 – 21 and days 1 - 4 of the lactation; no food consumption was recorded during the pairing period;
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of the scheduled necropsy (males); on day 5 post partum (females);
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia)
- Animals fasted: Yes
- How many animals: 5 males randomly selected from each group, 5 lactating females from each group;
- Parameters checked: Erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hemoglobin concentration distribution width, leukocyte count (total), differential leukocyte count, platelet count, reticulocytes, prothrombin time (= Thromboplastin time), activated partial thromboplastin time;

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of the scheduled necropsy (males); on day 5 post partum (females);
- Animals fasted: Yes
- How many animals: 5 males randomly selected from each group, 5 lactating females from each group;
- Parameters checked: Glucose, urea, creatinine, bilirubin (total), cholesterol (total), triglycerides, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl-transferase, bile acids, sodium, potassium, chloride, calcium, phosphorus, protein (total), albumin, globulin, albumin / globulin ratio

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at one time during the study (males two days before the scheduled sacrifice and females on day 3 post partum) following the daily dose administration;
- Dose groups that were examined: five P generation males and five P generation females randomly selected from each group;
- Battery of functions tested: Cage-side observations (faeces-balls, urine and posture as well as resistance to removal), hand-held observations (muscle tone, constitution, skin, pupil size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities), open field observations (level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine), reflexes (blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch,
responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex)), measurements / counts (hind limb / fore limb grip strength, rectal temperature); additionally, locomotor activity was measured quantitatively for the same animals (with an Activity Monitor AMS-0151 (FMI, Germany); activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes);

OTHER: Furthermore, an additional blood sample (0.5 mL) was collected into serum tubes for possible future measurement of the thyroid hormones triiodothyronine (T3) and thyroxine (T4). Serum samples were stored at ≤ -75°C. Any samples remaining at finalization of the study report were discarded.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
(For the parent animals, special attention was directed at the organs of the reproductive system.)
HISTOPATHOLOGY: Yes
(all gross lesions, testes, epididymides, prostate, seminal vesicles, ovaries, oviduct, vagina and uterus from all animals of the control and high-dose group, the remaining organs/tissues of 5 randomly selected males and females of the control and high-dose group, respectively, were examined histopathologically; special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure; histological examination of ovaries was carried out on the females that did not give birth; in addition, microscopic examination of the reproductive organs of all infertile males was made;)

Organ weights:
- all parental males: testes and epididymides (of all parental males);
- from 5 males and 5 females killed at the end of the study which were selected for hematology and clinical chemistry examination from each group: adrenal glands (weighed as pairs), brain, heart, kidneys (weighed as pairs), uterus (including cervix), prostate, liver, thymus, spleen, thyroid (after fixation), ovaries (weighed as pairs), seminal vesicles (inclusive coagulating gland);

Tissue preservation:
- all parental males: prostate, seminal vesicles with coagulating gland, testes (in Bouin’s fixative), epididymides (in Bouin’s fixative);
- all parental females: ovaries (with oviduct), uterus (with vagina);
- all males and females: gross lesions, brain, spinal chord (cervical, thoracic, lumbar), small and large intestines (incl. Peyer’s patches), stomach (forestomach and glandular stomach), liver, kidneys, adrenals, lymph nodes (axillary and mesenteric), urinary bladder, heart, thymus, thyroids and parathyroids, trachea and lungs, spleen, peripheral nerve (sciatic), bone marrow (femur);
- all males and females (only examined by histopathology in case of macroscopic findings indicative of potential toxicity): aorta, eyes with optic nerve and harderian gland, lacrimal gland, larynx, nasal cavity, esophagus, pituitary gland, femur with knee joint, mammary gland (male and female), pancreas, salivary glands – mandibular, sublingual, skeletal muscle, sternum with bone marrow, pharynx;
Statistics:
- food consumption, body and organ weights, clinical laboratory and reproduction data and macroscopical findings: means and standard deviations of various data were calculated; the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex; the Steel-test (many-one rank test) was applied instead of the
Dunnett-test when the data could not be assumed to follow a normal distribution; Fisher's exact-test was applied if the variables could be dichotomized without loss of information;
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were noted in controls or in animals treated at 7.5 and 25 mg/kg bw/day. At 75 mg/kg bw/day in males and females, bedding in mouth were observed from the end of the pre-pairing period or pairing period onwards until one day before necropsy. This was accompanied by slight salivation in some animals. This was considered to be a sign of discomfort and without toxicological relevance. No findings were noted at detailed weekly clinical observation.
Mortality:
no mortality observed
Description (incidence):
There were no unplanned deaths during the whole course of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test item-related effects on mean body weights and mean body weight gain were noted in males at 7.5 and 25 mg/kg bw/day. At 75 mg/kg bw/day, there was a slight decrease in mean body weight gain after treatment start during the first week of the pre-pairing period (23 g compared to 28 g in the control group). Afterwards no decrease was observed anymore. This slight effect could be test item-related but was considered to be not adverse. The overall differences in mean body weight gain at the dose levels of 0, 7.5, 25 and 75 mg/kg bw/day were: +17%, +15%, +17% and +16% during the pre-pairing period and +3%, +2%, +3% and +2% during the after pairing period (percentages refer to the body weight gain within the period). No test item-related effects on mean body weight and mean body weight gain were noted in females. The overall differences in mean body weight gain at the dose levels of 0, 7.5, 25 and 75 mg/kg bw/day were: +7%, +8%, +8% and +7% during the pre-pairing period, +50%, +52%, +53% and +50% during the gestation period and +5%, +3%, +6% and +4% during the lactation period (percentages refer to the body weight gain within the period).
Description (incidence and severity):
No test item-related effects on mean food consumption were noted in males and females.
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The assessment of the hematology data did not reveal any test item-related effects in males and females. Statistically significant differences (lower hemoglobin, MCHC, and eosinophil values in females at 7.5 mg/kg bw/day; lower eosinophil values in females and higher RDW levels in males at 25 mg/kg bw/day; higher HDW values in females at 75 mg/kg bw/day) showed either no dose-dependency or the values were in the range of the historical control data.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The assessment of the clinical biochemistry data did not reveal any test item-related effects in males and females. At 75 mg/kg bw/day, there was an increase in triglycerides and a decrease of total bilirubin in males and an increase in total cholesterol in females. The values of total bilirubin were within the historical control data. The alterations of triglycerides and cholesterol were considered to be of no toxicological relevance, since the differences to the control group were minor and were evident in one sex only. The same applies to the significantly higher ALP activities in females at 7.5 mg/kg bw/day, since there was no dose-dependency.
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related findings were noted during the functional observational battery in males and females at any dose level. One female treated at 7.5 mg/kg bw/day showed increased rearings. At 75 mg/kg bw/day, one female vocalized spontaneously when held in the hand. These isolated findings were considered to be incidental. Another female at 75 mg/kg bw/day showed salivation, which was already noted and discussed at the daily clinical investigation. The body temperature in males treated at 25 and 75 mg/kg bw/day was statistically significantly decreased (38.1°C and 37.6°C, respectively, compared to 38.6°C in the control group). Since these values were within the range of the historical control data (37.5 - 38.6°C), this finding was considered to be not test item-related. No such trend towards lower body temperatures was noted in females. Locomotor activity was not affected by the treatment with the test item in males or females at any dose level.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In males, no effects on organ weights were noted. In females, a dose-dependent and statistically significant increase in liver and kidney weight as well as liver and kidney to body and to organ weight ratio were noted at 25 and 75 mg/kg bw/day. This was considered compound-related, but not adverse, since there was no evidence for an impaired organ function by clinical chemistry, macroscopic examinations at necropsy and histopathology.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no gross lesions that could be attributed to treatment with the test item. All gross lesions recorded were considered to be within the range of normal background alterations. In males, incidental findings were a reddish or dark red discoloration of the pancreas and a yellowish and firm nodule in the pancreas. Additionally, two males showed following findings in the testes: at 25 mg/kg bw/day a hardening on the right side was noted, and at 75 mg/kg bw/day a reduction in size was present. However, those two males mated successfully. At microscopic examination of those two animals, the left testis of the male treated at 25 mg/kg bw/day was normal. In the male treated at 75 mg/kg bw/day, a moderate bilateral tubular degeneration/atrophy was noted at microscopic examination. Due to the low incidence and the lack of dose-dependency all these findings were considered to be incidental. In females, the most frequently incidental findings were a reddish or dark red discoloration of the ovaries and foci on or a reduction in size of the thymus, which was present in all dose levels without dose-dependency.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Dose descriptor:
NOAEL
Remarks:
(systemic)
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Critical effects observed:
not specified
Conclusions:
The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 75 mg/kg body weight/day, because no adverse effects were observed.
Executive summary:

The purpose of this OECD 422 study was to generate preliminary information on the possible health hazards likely to arise from repeated exposure to Bis(2 -ethylhexyl)amine. In addition, it provided information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

Di-(2-ethylhexyl)amine was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

The following dose levels were applied: 0 (control group), 7.5, 25, or 75 mg/kg body weight/day.

A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).

The following results were obtained:

Parent animals

- General Tolerability: There were no unplanned deaths during the course of this study. Clinical signs were limited to animals treated at 75 mg/kg bw/day and consisted of bedding in mouth, accompanied by slight salivation in some isolated animals. This was considered to be a sign of discomfort and without toxicological relevance.

- Food consumption: No effects on food consumption of males and females were observed at any dose level.

- Body weights: At 75 mg/kg bw/day, there was a slight decrease in mean body weight gain after treatment start (23 g compared to 28 g in the control group). This slight effect could be test item-related but was

considered to be not adverse. In females no effects on mean body weight and mean body weight gain were noted.

- Clinical laboratory investigations: The assessment of the hematology and clinical biochemistry data did not reveal any test item related effects in males and females.

- Reproduction and breeding data: Mean precoital time, fertility index and conception rate were not affected by the treatment with the test item. Mean number of litter size was similar in all groups and no effects on postnatal loss were observed at any dose level.

- Organ weights: Liver and kidney weights were increased in females at 25 and 75 mg/kg bw/day. These findings were considered compound-related, but not adverse, since there was no evidence for an impaired organ function by clinical chemistry and histopathology.

- Macroscopical findings and histopathological examinations: There were no test item-related findings noted at necropsy. All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

Litter data - F1 pups

- Findings at First Litter Check and during Lactation: No test item-related external findings were noted at first litter check.

- Pup Weights to Day 4 Post Partum: No effects on pup weight and pup weight gain were observed.

- Macroscopical Findings: At necropsy of pups, there were no abnormal findings.

Conclusion

The general NOAEL (No Observed Adverse Effect Level) was considered to be 75 mg/kg body weight/day.

The NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 75 mg/kg body weight /day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Available data is reliable and sufficient for assessment.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A GLP-compliant investigation of the toxicological effects resulting from repeated oral-gavage administration to rats was performed following OECD guideline 422 without deviations (BASF SE 2013; reliability score 1). The test substance was administered in corn oil as vehicle at dosages of 7.5, 25, and 75 mg/kg body weight/day, and controls received the vehicle only. The test substance was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum. At 75 mg/kg bw/day, bedding in mouth, accompanied by slight salivation in some isolated animals was noted. Furthermore, there was a slight decrease in mean body weight gain after treatment start in males, which was considered to be not adverse. Hematological and clinical chemistry examinations did not reveal any adverse effects. Increased liver and kidney weights were observed in females at 25 and 75 mg/kg bw/day. However, there were no hints for an impaired function of these organs by clinical chemistry and histopathology. All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age. No effects on reproduction and development were observed at any dose level. The general NOAEL was considered to be 75 mg/kg body weight/day.

Waiver Rep. dose, 90 d:

There is no sub-chronic (90 days) repeated dose study available for bis(2-ethylhexyl)amine.

In order to assess possible effects after repeated exposure, bis(2-ethylhexyl)amine was tested in a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD 422 (BASF SE, 2013). The substance was administered orally via gavage to groups of male and female Wistar rats at doses of 7.5, 25, and 75 mg/kg body weight/day. The exposure period for male rats was 28 days and female rats were exposed 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum. All observed effects were either isolated or considered to be not adverse. Therefore, the NOAEL for systemic toxicity was set at the highest dose tested, at 75 mg/kg body weight/day.

Additionally, the substance is classified as skin corr. cat. 1B.

Hence, since no further information is to be expected, the substance has corrosive properties, and with regard to animal welfare (reduction of animal testing), the conduct of a sub-chronic (90 days) study is deemed to be not necessary.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Key study (OECD 422)

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available data is reliable and the test substance has not to be classified under Regulation (EC) No.1272/2008.