Bis(2-ethylhexyl)amine

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

other: dose-range finder

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Principles of method if other than guideline:

The purpose of this study was to determine dose levels of Di-(2-ethylhexyl)amine suitable for use in a subsequent Reproduction/Developmental Toxicity Screening Test in the Han Wistar Rat.Groups of 4 male and female rats were treated by gavage for 14 days.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V.
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 269 - 364g, females: 203-236g
- Housing: individually
- Diet (e.g. ad libitum): ad lib. (Harlan Teklad 2018C (batch no. 80/11))
- Water (e.g. ad libitum): ad lib.
- Acclimation period: >= 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- 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: daily, homogeneity was maintained by constant stirring

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

all results were within 97 and 106.5% of the target concentration. Homogeneity differences were below 15%. Formulations were found to be stable when stored for 8 days at room temperature (maximum derivation: 1.6%)

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION
- Food consumption: recorded at weekly intervals per animal

OPHTHALMOSCOPIC EXAMINATION: No

BLOOD COLLECTION
- Time schedule for collection of blood: end of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (18h, water available ad lib.)
- How many animals: all (except high dose due to excessive mortality)

HAEMATOLOGY: Yes
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Protrhombin time
Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Aspartate aminotransferase
Alanine aminotransferase
Alkaline phosphatase
Gamma-glutamyl-transferase
Bile acids

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

Livers, kidneys, adrenals, spleen, and all macroscopic changes were preserved in neutral phosphate buffered 4% formaldehyde solution for possible microscopic examination
The organ weights were determined for Adrenal glands (weighed as pairs), Kidneys (weighed as pairs), Liver, Spleen

HISTOPATHOLOGY: No

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs observed at 300 mg/kg bw./day included salivation, bedding in mouth, decreased activity, ruffled fur, hunched posture, diarrhea, stiff gait, and reddish nasal secretion. Visible body weight loss and vocalization on touch was observed in one female. All these findings were considered to be test item-related.

Salivation and bedding in mouth were observed in males and females at 100 mg/kg bw./day. One male showed chromodacryorrhea.

At a dose level of 30 mg/kg bw/day, no clinical signs were recorded.

Mortality:

mortality observed, treatment-related

Description (incidence):

At a dose level of 300 mg/kg bw/day, male no. 9 and female no. 21 died spontaneously on day 9 or 8, respectively, and two females (nos. 22 and 24) were killed in extremis on day 8 of the treatment period. Due to the high mortality, dosing of the remaining animals was stopped on day 9 of the treatment period and these animals were sacrificed on day 10. The increased mortality was considered to be test item-related.

At a dose level of 30 and 100 mg/kg bw/day, all animals survived until planned necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At a dose level of 300 mg/kg bw/day, for males and females mean body weight gain was reduced after treatment start and after approximately one week a body weight loss was observed.

At a dose level of 100 mg/kg bw/day, mean body weight gain was reduced (statistically significant towards the end of the treatment period) during the whole treatment period (5% and -6% in males and females compared to 11% and 2% in the control group, respectively), resulting in slightly reduced mean body weights.

At a dose level of 30 mg/kg bw/day, mean body weight gain was similar to the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At a dose level of 300 mg/kg bw/day, mean food consumption was reduced by approximately 40% in males and females during treatment.

At a dose level of 100 mg/kg bw/day, mean food consumption was reduced by 12% (not statistically significantly) in males but increased by 4% in females during treatment.

At a dose level of 30 mg/kg bw/day, mean food consumption was slightly reduced (in males statistically significantly) during treatment. Since lower mean food consumption was already observed in the acclimatization period for this added dose group, this finding was considered to be a result of biological variability.

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 100 mg/kg bw/day in females, absolute and relative liver weights were slightly increased (+14% for relative liver weights compared to the control group).

No other alterations were noted. No organ weights have been assessed for the 300mg/kg group due to early euthanization.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg bw/day, male no. 9, which died spontaneously, had a distended stomach, liquid in the gut and enlarged adrenal glands. Also the three females (nos. 21, 22 and 24), which died spontaneously or were killed in extremis, had enlarged adrenal glands.

No other test item-related findings were noted at any dose level.

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

body weight and weight gain

Conclusions:

High dose animals showed body weight loss, diarrhea, and signs of pain and extreme discomfort, e.g., salivation, bedding in mouth, hunched posture, and vocalization, which are 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. The only systemic effect observed was an increase in liver weight and cholesterol, which is seen as a sign of metabolism of the substance and adaptive, but non-adverse. This view is supported by the absence of evidence for impaired liver function in the extensive examinations of the OECD 422 study. Based on these observations, dose levels of 7.5, 25 and 75 mg/kg bw/day were selected for the subsequent Reproduction/Developmental Toxicity Screening Test in the Han Wistar Rat.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl)amine

EC Number:

203-372-4

EC Name:

Bis(2-ethylhexyl)amine

Cas Number:

106-20-7

Molecular formula:

C16H35N

IUPAC Name:

bis(2-ethylhexyl)amine

Details on test material:

- Name of test material (as cited in study report): Di-(2-ethylhexyl)amine
- Physical state: Colourless, clear liquid
- Analytical purity: 98.5 g/100 g
- Lot/batch No.: 000STD77L0
- BASF test substance number: 11/0454-1
- Expiration date of the lot/batch: 11.11.2013
- Stability of Test Item in Vehicle: 8 days at room temperature
- Storage condition of test material: Room temperature (20 ± 5 °C)

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

7.5 mg/kg bw/day (nominal)

Remarks:

nominal conc. in vehicle

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (nominal)

Doses / concentrations 3

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.

Examinations

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;

Results and discussion

Results of examinations

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

Effect levels

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

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

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.