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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Aug 2013 - 09 Dec 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
21 Sep 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
Commission Regulation (EC) No 440/2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethyl-N,N-bis(2-ethylhexyl)hexylamine
EC Number:
217-461-0
EC Name:
2-ethyl-N,N-bis(2-ethylhexyl)hexylamine
Cas Number:
1860-26-0
Molecular formula:
C24H51N
IUPAC Name:
tris(2-ethylhexyl)amine
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source/batch No.of test material: BASF/O 3012
- Purity: 99.7 %
- Expiration date of the lot/batch: 04 July 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: guaranteed by the sponsor
- Stability under test conditions: room temperature for 7 days and 15 days in a refrigerator proved
- Analysis/Stability of the test substance in the Ground Kliba maintenance diet: analytically tested at the start and end of administration period (3 samples for low and high dose preparations and 1 sample for the mid dose preparation)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: mixed with diet (for about 10 minutes in a laboratory mixer)

FORM AS APPLIED IN THE TEST mixture of test material with diet

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain as described in the report: Crl:WI(Han)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: 42 +/- 1 days
- Mean body weight at test initiation: males, about 188 g; females, about 148 g
- Housing: five /cage, in H-Temp (PSU) cages [TECNIPLAST, Hohenpeißenberg, Germany; floor area 800 cm2]
- Diet: ground Kliba mouse/rat maintenance diet “GLP”, meal (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 9 days

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

ANALYSIS OF FOOD, WATER, BEDDING
- The food used in the study was assayed for chemical and microbiological contaminants.
- The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory.
- The bedding and the enrichment are regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION:
For each concentration, the test substance was weighted out and mixed with a small amount of food. In order to obtain the desired concentrations, these premixes were added to the corresponding amounts of food, depending on test group. Mixing was carried out for about 10 minutes in a laboratory mixer.


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, as a part of this study.
Homogeneity was verified in 3 samples in the highest and lowest concentrations (was used as a concentration control at the same time) at the beginning of the administration period; additional concentration control analyses were verified in 1 sample of the mid concentration at the same time. Additional concentration control analyses were performed for all concentrations towards the end of the administration period.
Duration of treatment / exposure:
90 day
Frequency of treatment:
once daily, seven days/week, during 3 months
Doses / concentrationsopen allclose all
Dose / conc.:
300 ppm
Remarks:
equivalent to 19 mg/kg bw/d in males and 21 mg/kg bw/d in females
Dose / conc.:
2 000 ppm
Remarks:
equivalent to 131 mg/kg bw/d in males and 146 mg/kg bw/d in females
Dose / conc.:
12 000 ppm
Remarks:
equivalent to 822 mg/kg bw/d in males and 885 mg/kg bw/d in females
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: after request of the sponsor
- Fasting period before blood sampling for clinical biochemistry: at least 16 hours
Positive control:
-

Examinations

Observations and examinations performed and frequency:
MORTALITY AND CLINICAL OBSERVATIONS
Check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. Moribund animals were sacrificed in extremis and were subjected to necropsy.

DETAILED CLINICAL OBSERVATION
Detailed clinical observations 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. For the purpose of these examinations, the rats were transferred into a standard arena (50 x 37.5 x 25 cm). The following parameters were examined: abnormal behaviour during handled, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmia, appearance and consistency of faeces, urine and pupil size.

BODY WEIGHT
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION
Food consumption was determined weekly for each cage. The mean daily intake of test substance (group means) was calculated based upon individual values for body weight and mean food consumption per cage.

WATER CONSUMPTION
Daily visual check of the water bottles within the scope of general observation was done.

NEUROBEHAVIOURAL EXAMINATION, FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
A functional observational battery (FOB) was performed in all animals at the end of the administration period starting at about 10:00 h. At least one hour before the start of the FOB the animals were transferred to single-animal polycarbonate cages. Drinking water was provided ad libitum, but no food was offered during the measurements. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

- HOME CAGE OBSERVATION:
The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behaviour of the animals. Following parameters were considered: posture, tremor, convulsions, abnormal movements, gait abnormalities

- OPEN FIELD OBSERVATION
The animals were transferred into a standard arena (50 x 50 cm with sides of 25 cm high) and were observed for at least 2 minutes. Following parameters were examined: behaviour when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypes, impairment of gait, activity/arousal level, faeces (number of faecal pellets/appearance/consistency) within two minutes, urine (amout/color) within two minutes, number of rearing within two minutes.

- SENSORY MOTOR TESTS/REFLEX TESTS
The animals were removed from the open field and subjected to following sensorimotor or reflex tests:
1. approach response
2. touch response
3. vision ("visual placing response")
4. pupillary reflex
5. pinna reflex
6. audition ("startle response")
7. coordination of movements ("righting response")
8. behaviour during "handling"
9. vocalization
10. pain perception ("tail pinch")
11. grip strength of forelimbs
12. grip strength of hind limbs
13. landing foot-splay test
14. other findings

MEASUREMENT OF MOTOR ACTIVITY (MA)
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts was counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal. The program requires a file name for the measured data to be stored. This name consists of the reference number and a serial number.

OPHTHALMOLOGICAL EXAMINATION
The eyes of all animals were examined prior to the start of the administration period. At the end of the administration period (study day 91), the eyes of animals in the control group and the high dose group [12000 ppm bw/d] were examined for any changes using an ophthalmoscope after administration of a mydriatic.

ESTROUS CYCLE DETERMINATION
Estrous cycle length and normality were evaluated daily for all female animals for a minimum of 3 weeks prior to necropsy.

HEMATOLOGY AND CLINICAL CHEMISTRY
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence.
The following examinations were carried out in all animals per test group and sex at the end of the administration period.
Leukocyte count (WBC), Erythrocyte count (RBC) , Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET), Prothrombin time (Hepato Quick’s test) (HQT)

Following clinical chemical parameters were examined in 10 animals per test group and sex:
Sodium, potassium, glucose, chloride, calcium, inorganic phosphate, urea, creatinine, triglycerides, total protein, total bilirubin, albumin, globulins, Cholesterol

Following enzymes were considered:
Alkaline phosphatase (AP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-Glutamyltransferase (GGT).

URINALYSIS
The dry chemical reactions on test strips used to determine urine constituents semiquantitatively were evaluated with a reflection photometer.
Volume, colour, turbidity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity and sediment were measured/examined.

SPERM PARAMETERS
Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals.
Sperm motility, Sperm morphologym Sperm head count (cauda epididymis), Sperm head count (testis) were evaluated.
Sacrifice and pathology:
Prior sacrifice, the anesthetized animals were weighed. They were then sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS
Following organs were weighed: Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles, Spleen, Testes, Thymus, Thyroid glands, Uterus with cervix

ORGAN/TISSUE FIXATION [FORMALDEHYDE 4% SOL. OR MODIFIED DAVIDSON'S SOL.]
Samples from following organs/tissues as well as gross lesions were collected and fixed in neutral 4% buffered formaldehyde for further histopathological examinations:
All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenumj, Epididymides, left, Esophagus, Eyes with optic nerve, Female mammary gland, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes (mesenteric and axillary lymph nodes), Ovaries, Pancreas, Parathyroid glands, Peyers'patches, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Stomach (forestomach and glandular stomach), Testis, left, Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina

HISTOPATHOLOGY
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to :
All gross lesions; Adrenal gland; Aorta; Bone marrow (femur); Brain; Cecum; CervixCoagulating glands; Colon; Duodenum; Epididymis, left; Esophagus; Eyes with optic nerve; Female mammary gland; Heart; Ileum; Jejunum; Kidney; Liver; Lung; Lymph nodes, mesenteric and axillary lymph nodes; Ovaries; Pancreas; Parathyroid glands; Peyer’s patches, Pituitary gland; Prostate; Rectum; Salivary glands (mandibular and sublingual glands); Sciatic nerve; Seminal vesicles; Skin; Spinal cord (cervical, thoracic and lumbar cord); Spleen; Stomach (forestomach and glandular stomach); Testis, left; Thymus; Thyroid glands; Trachea; Urinary bladder; Uterus; Vagina.
Statistics:
The tests used for the statistical assessment of the results for the different parameters considered can be summarized as follows:
- Body weight, body weight change,: Comparison of each group with control group using DUNNETT's test (two-sided) for the hypothesis of equal means;
- Feces, rearing, grip strength forelimbs, grip strength hindlimbs, foot-splay test, motor activity, estrous cycle: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.

Statistics of clinical pathology:
Blood parameters: For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two- sided) for the hypothesis of equal medians

For parameters with unidirectional changes:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians;* for p ≤ 0.05, ** for p ≤ 0.01
Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians; * for p ≤ 0.05, ** for p ≤ 0.01.
Spermanalysis parameters: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians; If only control and one dose group are measured, WILCOXON-test (one- sided) without adjustment were used. For the percentage of abnormal sperms (ABNORMAL6_C) values < 6% were set to 6 % (cut off 6%); * for p ≤ 0.05, ** for p ≤ 0.01

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No changes were observed in male and female animals of test groups 1, 2 and 3 (300, 2000 and 12000 ppm).
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely in the present study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No significant changes to the control values were observed in male and female animals of test groups 1, 2 and 3 (300, 2000 and 12000 ppm). However, although not significantly altered, mean body weight was lower in male animals of test group 3 (12000 ppm) by -6.2% on study day 91. In addition, the mean body weight change value was significantly lower (not significantly altered) in male animals by -9.7% on study day 91.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In table 1 the intake of the test substance is shown.
During numerous time intervals, food consumption in female animals of test group 1-3 (300, 2000 and 12000 ppm) was slightly lower when compared to the control group. As no dose-response relationship occurred the changes were assessed to be incidental.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related findings were reported.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the administration period in females of test group 3 (12000 ppm), total white blood cell counts (WBC), absolute neutrophil, lymphocyte and monocyte cell counts as well as relative monocyte cell counts were increased. In these individuals absolute basophil cell counts were also higher compared to controls, but the mean was still within the historical control range (absolute basophil cell counts 0-0.05 Giga/L; Therefore, this alteration was regarded as incidental and not treatment-related.
In females of test group 2 (2000 ppm) absolute neutrophil and monocyte cell counts were higher compared to controls, but the means of these parameters as well as the total white blood cell count (WBC) mean in these individuals were within historical control ranges (absolute neutrophil cell counts 0.37-0.93 Giga/L; absolute monocyte cell counts 0.04-0.10 Giga/L, WBC 2.80-4.42 Giga/L.
Therefore, the changes in females of test group 2 (2000 ppm) were regarded as incidental and not treatment-related. Prothrombin time (HQT, Hepatoquick’s test) could not be measured in females at the end of the study due to a technical failure of the instrument. However, this fact did not influence the overall validity of the study.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in rats of both sexes of test group 3 (12000 ppm) alanine aminotransferase (ALT) activities were increased. Additionally, in females of this test group gamma-glutamyl transferase (GGT) activities were slightly increased. In females of test group 2 (2000 ppm) GGT activities were already higher compared to controls, but the mean was within the historical control range (GGT 0-14 nkat/L); and, therefore, GGT increase in females of test group 2 (2000 ppm) was regarded as incidental and not treatment-related.
Albumin levels were decreased in females of test group 3 (12000 ppm).
Cholesterol levels were higher compared to controls in rats of both sexes of test groups 2 and 3 (2000 and 12000 ppm) and additionally in males of test group 1 (300 ppm). However, in both sexes cholesterol levels were not changed dose-dependently regarding means. In females, the means were within the historical control range and those in males were marginally above the range (cholesterol, females 1.06-2.03 mmol/L, males 1.51-2.23 mmol/L. In test groups 1 and 2 (300 and 2000 ppm) only cholesterol values were altered. Because of the lacking dose-dependency, the cholesterol changes were regarded as incidental and not treatment-related (ECETOC Technical Report No 85, 2002).
Alkaline phosphatase (ALP) activities were decreased in females of test group 1 (300 ppm), but this decrease was not dose-dependent. Urea levels were increased in females of test group 3 (12000 ppm) and calcium levels were higher in males of the same test group. The means of both parameters were within historical control ranges (urea levels in females 6.07- 9.03 mmol/L; calcium males 2.35-2.76 mmol/L. Therefore, the mentioned parameter changes were regarded as incidental and not treatment-related.

Total bilirubin levels were decreased in rats of both sexes of test groups 2 and 3 (2000 and 12000 ppm). Without any signs of anemia the cause for a decrease of bilirubin levels was most probably an increased conjugation rate in the liver followed by an increased excretion via bile. No pathophysiological effect can be correlated with this lower bilirubin level. Therefore, the lower total bilirubin levels in rats of both sexes of test groups 2 and 3 (2000 and 12000 ppm) were regarded as treatment-related but not adverse.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to rather have been incidental.
Referring to home cage observation, open field observations,sensorimotor tests and reflexes and quantitative parameters, no substance-related findings were observed.
Regarding the single intervals and overall motor activity, no treatment-related deviations were observed.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
When compared with control group 0 (set to 100%), mean absolute weights were significantly changed, as shown in table 2.
All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

When compared with control group 0 (set to 100%), mean relative organ weights were significantly changed, as shown in table 3.
All other mean relative weight parameters did not show significant differences when compared to the control group 0.

The significantly increased weights in ovaries and spleen of females of test group 2 (2000 ppm) and test group 3 (12000 ppm) were regarded to be treatment-related. The increases in absolute liver weight in females of all test groups and in relative liver weight in test group 2 (2000 ppm) and test group 3 (12000 ppm) females were most likely test substance-related.
The absolute weight increase in kidneys in females of test group 2 (2000 ppm) was not regarded to be treatment-related due to a missing dose-response relationship and unchanged relative kidneys weight.

The decrease in absolute brain weight in test group 3 (12000 ppm) males was regarded to be not related to treatment as the relative brain weight was not significantly altered and no histopathologic findings were observed.
The significant increase in kidney, liver and testes weights in males of test group 3 (12000 ppm) were regarded to be due to a slight decrease in terminal body weight that was not significant. The changes were therefore regarded to be incidental and not related to treatment.
The increase in relative spleen weight in males of test group 2 (2000 ppm) and test group 3 (12000 ppm) was regarded to be incidental due to a missing histopathologic correlate and the organ weights were still within historical control data .
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Gross findings were observed in females with incidences according to the table 4.
The enlargements of the above mentioned organs were regarded to be treatment-related, with the exception of the renal lymph node in test group 2 (2000 ppm).
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.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In general, females were more severely affected than males.
In several organs were mainly histiocytic cell infiltrates accompanied by less granulocytes. The infiltrating histiocytes revealed a finely vacuolated cytoplasm.

Ovaries - findings shown in table 6:
The ovaries revealed mainly within corpora lutea and to a much lower extent in the interstitial glands and interstitium an infiltration of histiocytes and lower numbers of granulocytes, a foamy change, focal fibrosis and a single animal showed multinucleated giant cells. This represents a granulomatous inflammation that showed a dose-response relationship and was regarded to be treatment-related.

Small intestine (exemplarily jejunum) - findings shown in table 7:
Within the tips of the villi of the small intestine there were mainly histiocytic infiltrates. Same animals showed similar infiltrates also in the muscular wall, often adjacent to the Peyer’s patches. Here, higher numbers of granulocytes were observed. In test groups 2 (2000 ppm) animals the infiltrates were often only found within the intestinal wall. The jejunum was the most severely affected part in the small intestine; therefore, the incidences of the jejunum are shown in the table exemplarily for the rest of the digestive tract. In females all regions of the small intestine were affected, whereas in males only the duodenum and jejunum showed findings.
One female of test group 3 (12000 ppm) revealed also histiocytic infiltrates in the wall of the glandular stomach and in Peyer’s patch. These findings were regarded to be treatment- related.

Mesenteric lymph nodes - findings shown in table 8:
In the mesenteric lymph nodes there were histiocytic cell infiltrates, occasionally intermingled with multinucleated giant cells and additional granulocytic infiltrates. Some animals showed marked fibrosis that often extended into the surrounding fat tissue or focal mineralization. Mainly in test group 3 (12000 ppm) the infiltrating granulocytes were forming microabscesses. These findings represent a strong granulomatous inflammation with foreign body reaction (multinucleated giant cells) that show a dose-response relationship and were regarded to be related to treatment. In the most severely affected animals, the granulomatous inflammation and fibrosis replaced the organ compartments leading to a secondary depletion of lymphoid tissue.
The renal lymph nodes of three females of test group 3 (12000 ppm) showed similar findings which correlated to the macroscopic observed enlargement of these lymph nodes.

Bone marrow - findings shown in table 9:
In the femoral bone marrow of female animals of test group 3 (12000 ppm) there was an increase in myeloid cells. This was regarded to be a consequence to the inflammatory reaction described for several organs and represent an increase in white blood cell production. This finding was regarded to be treatment-related.

Spleen - findings shown in table 10:
In the spleen there was an increase in red blood cell production in females. This was regarded to be most likely a secondary consequence to the findings in the bone marrow (shift of myeloid/erythroid ratio) and regarded to be treatment related. In males there was a minimal to slight increase in few animals of all test groups with no difference between control and treated animals. In males this was therefore not regarded to be a treatment related finding but incidental.
Other effects:
no effects observed
Description (incidence and severity):
No test substance-related effects on estrous cycle length and the number of cycles were obtained.
Concerning the motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as the sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.
Details on results:
The codes that are used at finding levels are described in a grading system that takes into consideration either the severity or the number or the size of a microscopic finding.
This grading system is shown in table 5.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: substance-related adverse signs of systemic toxicity at a concentration of 2000 ppm in male and of 300 ppm in female rats
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
female
Basis for effect level:
other: A NOAEL in female Wistar rats was not achieved and below 300 ppm (<21 mg/kg bw/d) taking findings in ovaries and mesenteric lymph nodes into account.
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Intake of the test substance

 Test group

Concentration in the vehicle (ppm)

Mean daily test-substance intake (mg/kg bw/d)

Males

Females

1

300

19

21

2

2000

131

146

3

12000

822

885

Table 2: Absolute organ weights of selected organs with significant changes

 

Male animals

Female animals

Test group (ppm)

1

(300)

2

(2000)

3

(12000)

1

(300)

2

(2000)

3

(12000)

Brain

98%

101%

96%**

 

 

 

Kidneys

 

 

 

104%

113%**

106%

Liver

 

 

 

107%*

114%**

115%**

Ovaries

 

 

 

114%

162%**

235%**

Spleen

 

 

 

107%

122%**

151%**

*: p ≤ 0.05, **: p ≤ 0.01

Table 3: Relative organ weights of selected organs with significant changes

 

Male animals

Female animals

Test group (ppm)

1

(300)

2

(2000)

3

(12000)

1

(300)

2

(2000)

3

(12000)

Epididymides

98%

104%

110%*

 

 

 

Kidneys

97%

102%

110%**

 

 

 

Liver

98%

100%

107%**

104%

108%*

112%**

Ovaries

 

 

 

110%

154%**

229%**

Spleen

100%

113%*

121%**

103%

116%*

147%**

Testes

97%

106%

111%**

 

 

 

*: p ≤ 0.05, **: p ≤ 0.01

Table 4: Gross lesion observed in females

 

Female animals

Test group (ppm)

0

(0)

1

(300)

2

(2000)

3

(12000)

No. of animals

10

10

10

10

Ovaries, enlarged

0

0

3

9

Mesenteric lymph nodes, enlarged

0

0

1

4

Renal lymph nodes, enlarged

0

0

1

3

Spleen, enlarged

0

0

0

2

Table 5: Codes used for findings (grading system):

 

Severity

Number

Size

Grade 1

Minimal

Very few

Very small

Grade 2

Slight

Few

Small

Grade 3

Moderate

Moderate number

Moderate size

Grade 4

Marked; severe

Many

Large

Grade 5

Massive; extreme

Extensive number

Extensive size

Whenever a grading was not used, the microscopic finding was indicated to be present (P).

Table 6: Histopathological findings in the ovaries

 

Female animals

Test group (ppm)

0

(0)

1

(300)

2

(2000)

3

(12000)

No. of animals

10

10

10

10

Infiltrates histiocytic/mixed cells

0

2

10

10

-Grade1

 

2

 

 

-Grade2

 

 

4

1

-Grade3

 

 

6

5

-Grade4

 

 

 

4

Infiltrates multinucleated giant cells

0

0

0

1

-Grade1

 

 

 

1

Foamy change

0

4

10

10

-Grade1

 

2

2

 

-Grade2

 

2

6

3

-Grade3

 

 

2

5

-Grade4

 

 

 

2

Fibrosis, (multi)focal

0

0

3

6

-Grade1

 

 

2

3

-Grade2

 

 

1

3

Table 7: Histopathological findings in the small intestine (exemplarily jejunum)

 

Male animals

Female animals

Test group (ppm)

0

(0)

1

(300)

2

(2000)

3

(12000)

0

(0)

1

(300)

2

(2000)

3

(12000

)

No. of animals

10

10

10

10

10

10

10

10

Infiltrates histiocytic/ mixed

cells

0

0

1

7

0

0

8

9

-Grade1

 

 

 

2

 

 

6

4

-Grade2

 

 

1

5

 

 

2

3

-Grade3

 

 

 

 

 

 

 

2

Table 8: Histopathological findings in the mesenteric lymph nodes

 

Male animals

Female animals

Test group (ppm)

0

(0)

1

(300)

2

(2000)

3

(12000)

0

(0)

1

(300)

2

(2000)

3

(12000)

No. of animals

10

10

10

10

10

10

10

10

Infiltrates histiocytic/mixed

cell

0

0

8

10

0

1

9

9

-      Grade1

 

 

6

2

 

1

4

 

-      Grade2

 

 

1

3

 

 

4

3

-      Grade3

 

 

1

4

 

 

1

5

-      Grade4

 

 

 

1

 

 

 

1

Infiltrates multi-

nucleated giant cells

0

0

5

5

0

0

6

4

-      Grade1

 

 

5

1

 

 

4

1

-      Grade2

 

 

 

4

 

 

1

2

-      Grade3

 

 

 

 

 

 

1

1

Fibrosis, (multi)focal

0

0

1

3

0

0

4

4

-      Grade1

 

 

1

 

 

 

 

 

-      Grade2

 

 

 

3

 

 

2

 

-      Grade3

 

 

 

 

 

 

2

3

-      Grade5

 

 

 

 

 

 

 

1

Mineralization, (multi)focal

0

0

0

4

0

0

0

1

-      Grade1

 

 

 

2

 

 

 

1

-      Grade2

 

 

 

2

 

 

 

 

Depletion, lymphoid

0

0

0

0

0

0

1

4

-      Grade2

 

 

 

 

 

 

1

1

-      Grade3

 

 

 

 

 

 

 

2

-      Grade4

 

 

 

 

 

 

 

1

Table 9: Histopathological findings in the bone marrow of female animals

 

Female animals

Test group (ppm)

0

(0)

1

(300)

2

(2000)

3

(12000)

No. of animals

10

10

10

10

Hyperplasia myeloid

0

0

0

3

-Grade2

 

 

 

1

-Grade3

 

 

 

1

-Grade4

 

 

 

1

Table 10: Histopathological findings in the spleen of female animals

 

Female animals

Test group (ppm)

0

(0)

1

(300)

2

(2000)

3

(12000)

No. of animals

10

10

10

10

Hematopoiesis, extramedullary

0

0

5

8

·        Grade1

 

 

3

5

·        Grade2

 

 

2

3

Applicant's summary and conclusion

Conclusions:
The oral administration of the test substance via the diet to male and female Wistar rats for 3 months revealed test substance-related adverse signs of systemic toxicity at concentrations of 2000 ppm (equivalent to 131 mg/kg bw/d) in male and of 300 ppm (equivalent to 21 mg/kg bw/d) in female Wistar rats.
Therefore, under the conditions of the present study, the no observed adverse effect level (NOAEL) was 300 ppm in male (equivalent to 19 mg/kg bw/d) Wistar rats. A NOAEL in female Wistar rats was not achieved and below 300 ppm (<21 mg/kg bw/d) taking findings in ovaries and mesenteric lymph nodes into account.
Executive summary:

SUMMARY

METHODS

Tris-(2-ethylhexyl)amine was administered via the diet to groups of 10 male and 10 female Wistar rats at concentrations of 0 (test group 0), 300 (test group 1), 2000 (test group 2) and 12000 ppm (test group 3) over a period of 3 months.

OBSERVATIONS

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Ophthalmological examinations were performed before the beginning and at the end of the administration period. For at least 3 weeks an estrous cycle determination was performed.

Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period.

Clinicochemical and hematological examinations as well as urinalyses were performed towards the end of the administration period.

After the administration period all animals were sacrificed and assessed by gross pathology, followed by histopathological examinations.

RESULTS

Analytics

The various analyses confirmed

• the stability of the test-substance preparations for a period of 7 days at room temperature and 15 days in a refrigerator,

• the homogeneous distribution of the test substance in the vehicle,

• the correctness of the prepared concentrations.

Findings

The following test substance-related, adverse findings were noted:

Test group 3: 12000 ppm (822 mg/kg bw/d in males and 885 mg/kg bw/d in females)

Clinical Examinations

• Although not significantly altered, mean body weight was lower in male animals by -6.2% on study day 91.

• Although not significantly altered, mean body weight change value was significantly lower in male animals by -9.7% on study day 91.

Clinical Pathology

• Increased alanine aminotransferase (ALT) activities in rats of both sexes

• Increased γ-glutamyl transferase (GGT) activities in females

• Increased total white blood cell counts, absolute neutrophil, lymphocyte and monocyte cell counts as well as relative monocyte cell counts in females

Pathology

• Macroscopically enlarged ovaries in nine females

• Macroscopically enlarged spleens in two females

• Macroscopically enlarged mesenteric lymph nodes in four females

• Macroscopically enlarged renal lymph nodes in three females corresponding to granulomatous inflammation (histiocytic/mixed cell infiltrates, fibrosis, multinucleated giant cells)

• Up to severe granulomatous inflammation of ovaries (histiocytic/mixed cell infiltrates, fibrosis, multinucleated giant cells, foamy change) in all females

• Up to moderate histiocytic/mixed cell infiltrates in the villi or wall of the small intestine of seven males and nine females

• Up to severe granulomatous inflammation of mesenteric lymph nodes (histiocytic/mixed cell infiltrates, fibrosis, multinucleated giant cells) in all males and nine females

Test group 2: 2000 ppm (131 mg/kg bw/d in males and 146 mg/kg bw/d in females)

Clinical Examinations and Clinical Pathology

• No treatment-related, adverse effects were observed.

Pathology

• Macroscopically enlarged ovaries in three females

• Macroscopically enlarged mesenteric lymph nodes in one female

• Up to moderate granulomatous inflammation of ovaries (histiocytic/mixed cell infiltrates, fibrosis, multinucleated giant cells, foamy change) in all females

• Up to slight histiocytic/mixed cell infiltrates in the villi or wall of the small intestine of one male and eight females

Test group 1: 300 ppm (19 mg/kg bw/d in males and 21 mg/kg bw/d in females)

Clinical Examinations, Clinical Pathology and

• No treatment-related, adverse effects were observed.

Pathology

• Minimal histiocytic/mixed cell infiltrates in the ovaries of two females

• Minimal histiocytic/mixed cell infiltrates in the mesenteric lymph nodes of one female

CONCLUSION

The oral administration of Tris-(2-ethylhexyl)amine via the diet to male and female Wistar rats for 3 months revealed test substance-related adverse signs of systemic toxicity at a concentration of 2000 ppm in male and of 300 ppm in female Wistar rats. Therefore, under the conditions of the present study, the no observed adverse effect level (NOAEL) was 300 ppm in male (19 mg/kg bw/d) Wistar rats. A NOAEL in female Wistar rats was not achieved and below 300 ppm (<21 mg/kg bw/d) taking findings in ovaries and mesenteric lymph nodes into account.