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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Oct 2009 - 08 Nov 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study conducted in compliance with GLP regulations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tris-(2-ethylhexyl)amin O 2446
- IUPAC name: 2-Ethyl-N,N-bis(2-ethylhexyl)-hexylamine
- Physical state: liquid / colorless, clear
- Analytical purity: 99.7 corrected area-%, corrected for the water content (BASF Analytical Report, study code 09L00245)
- Purity test date: 27 Oct 2009
- Lot/batch No.: Behälter 407 v. 16.07.2009
- Stability under test conditions: The stability under storage conditions was confirmed by reanalysis.
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Wistar, Crl:WI(Han)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11 - 13 weeks old
- Weight at study initiation: male animals: 281.2 g - 307.2 g; female animals: 180.1 g - 211.2 g
- Housing: individually in Makrolon type M III cages, except during overnight matings, when male and female mating partners were housed together.
- Diet (ad libitum): Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum
- Acclimation period: about 6 days

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

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Test diets were prepared five times during the study. The maximum period for which each test diet was stored in a refrigerator was 21 days. The maximum period for which each test diet was fed was 7 days.
- Storage temperature of food: refrigerator

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The following analytical verifications of the stability of the test substance in the diet were carried out prior to the start of the study or during the study:
• 14 days at room temperature
• 42 days storage in refrigerator
• 15 days storage in refrigerator and subsequently 0, 7, 14 and 27 days at room temperature
Homogeneity and concentration control analyses were carried out at the beginning of the premating period and during gestation period.

All measured values for Tris-(2-ethylhexyl)amin O 2446 were in the expected range of the target concentrations (90 - 110 %), demonstrating the correctness of the diet preparations.
Duration of treatment / exposure:
Females: 51 or 53 days
Males: 31 days
Frequency of treatment:
continuously in the diet until 16 hours before sacrifice
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1500, 4000, and 12000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
125, 327, and 900 mg/kg bw/d
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
about 105, 277 and 815 mg/kg bw/d in males; about 103, 271, 770 mg/kg bw/d in non-pregnant females; about 121, 306 and 918 mg/kg bw/d in pregnant females; about 173, 455, 1098 mg/kg bw/d in lactating females
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed clinical observation (DCO) was performed in all animals once before the first administration and thereafter at weekly intervals.
- For observation, the animals were removed from their cages by the investigator and placed in a standard arena for at least 20 seconds/animal (50 x 37.5 cm wide, with side borders which are 25 cm high). The following parameters listed were assessed:
1. Abnormal behavior in handling
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos
15. Assessment of the feces discharged during the examination (appearance/consistency)
16. Assessment of the urine discharged during the examination
17. Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: once a week
- The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day after parturition (PND 1) and on PND 4.
Body weight was not determined in females showing no positive evidence of sperm during mating and gestation periods or in females without litter during lactation period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male F0 animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined for gestation days (GD) 0 - 7, 7 - 14 and 14 - 20.
• Food consumption of F0 females, which gave birth to a litter was determined for PND 1- 4.
Food consumption was not determined in females without positive evidence of sperm during mating and gestation periods and in females without litter during lactation period.

The intake of test substance was calculated from the amount of food consumed and expressed in mg/kg body weight per day.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological examinations were carried out on study days 31 (males) and 51 (females).
- Anaesthetic used for blood collection: Yes (isoflurane (Isoba®, Essex GmbH Munich, Germany))
- Animals fasted: Yes
- How many animals: 5 animals per test group and sex
- Parameters examined: 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, Prothrombin time (Hepato Quick’s test) (HQT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Clinicochemical examinations were carried out on study days 31 (males) and 51 (females).
- Anaesthetic used for blood collection: Yes (isoflurane (Isoba®, Essex GmbH Munich, Germany))
- Animals fasted: Yes
- How many animals: 5 animals per test group and sex
- Parameters examined: Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase), Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase), Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase), γ-Glutamyltransferase (GGT) (γ -glutamyl) peptide: aminoacid-γ- glutamyl-transferase), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Magnesium (MG)

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis were carried out from 5 male and 5 female animals (with litter) per group, selected by chance, on study day 28 (males) and 46 (females).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, Turbidity, Volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
On study day 28, a functional observational battery and motor activity measurement were carried out in five male animals per group.
On study day 50, a functional observational battery and motor activity measurement was carried out in five female animals (with litter) per group.
- How many animals: five animals per sex and group (females only with litter)
- Battery of functions tested: sensory activity / grip strength / motor activity
- Home cage observations:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Impairment of gait
6. Other findings
- Open field observations:
1. Behavior when removed from cage
2. Fur
3. Skin
4. Salivation
5. Nose discharge
6. Lacrimation
7. Eyes/pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/stereotypy
14. Impairment of gait
15. Activity/arousal level
16. Feces excreted during the observation period
17. Urine excreted during the observation period
18. Number of rearings
- Sensory motor tests/Reflexes:
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. Behavior during “handling”
9. Vocalization
10. Pain perception (“tail pinch”)
11. Other findings
12. Grip strength of forelimbs and hindlimbs
13. Landing foot-splay test
- Motor activity measurement (MA): The examinations were performed using the Multi-Varimex system supplied by Columbus Instruments Int. Corp., Ohio, U.S.A.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Ovaries
10. Pituitary gland
11. Prostate
12. Seminal vesicles with coagulation glands
13. Spleen
14. Testes
15. Thymus
16. Thyroid glands
17. Uterus with cervix

HISTOPATHOLOGY: Yes
The following organs or tissues were fixed in 4% formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Coagulation glands
8. Colon
9. Duodenum
10. Epididymides (modified Davidson’s solution)
11. Esophagus
12. Eyes with optic nerve
13. Female mammary gland
14. Femur with knee joint
15. Heart
16. Ileum
17. Jejunum (with Peyer’s patches)
18. Kidneys
19. Larynx
20. Liver
21. Lungs
22. Lymph nodes (mesenteric and axillary lymph nodes)
23. Nose (nasal cavity)
24. Ovaries (modified Davidson’s solution)
25. Oviducts
26. Pancreas
27. Pharynx
28. Pituitary gland
29. Prostate
30. Rectum
31. Salivary glands (mandibular and sublingual glands)
32. Seminal vesicle with coagulation glands
33. Sciatic nerve
34. Skeletal muscle
35. Skin
36. Spinal cord (cervical, thoracic and lumbar cords)
37. Spleen
38. Sternum with marrow
39. Stomach (forestomach and glandular stomach)
40. Testes (modified Davidson’s solution)
41. Thymus
42. Thyroid glands/parathyroid glands
43. Trachea
44. Urinary bladder
45. Uterus
46. Vagina
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings. All hematoxylin-eosin embedded tissues from the control and high-dose group animals were assessed (in most cases from 5 animals/sex/group).
Statistics:
- Food consumption, body weight and body weight change: DUNNETT-test (two-sided)
- Faeces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity: KRUSKAL-WALLIS test (two-sided), WILCOXON test (two-sided)
- Clinical pathology parameters, urine volume, urine specific gravity: KRUSKAL-WALLIS test (two-sided)
- Urinalysis, except color, turbidity, volume and specific gravity: FISHER's exact test
- Organ weight parameters: KRUSKAL-WALLIS test (two-sided), WILCOXON-test (two-sided)

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
There were no test substance-related or spontaneous mortalities in any of the groups.
No clinical signs or changes of general behaviour, which may be attributed to the test substance, were detected in any male or female F0 generation parental animals during the whole study including gestation and lactation periods.
One sperm positive control female, one sperm positive low-dose female, three sperm positive mid-dose, and two high-dose females did not deliver F1 pups.

BODY WEIGHT AND WEIGHT GAIN
Body weights of the high-dose females (12000 ppm) were statistically significantly lower during gestation (9 % below control on GD 20) and during lactation (PND 1 weight 8 % and PND 4 weight 7 % below control). Body weight gain of the high-dose females was statistically significantly lower than control during gestation (GD 7 - 20 up to about 37 %, GD 0 - 20 about 25 %). Body weights and body weight gain of all treated males were unchanged throughout the study as were body weight/ body weight gain of the low- and mid-dose females. This statement includes the statistically significantly increased body weights of the low- and middose females during premating week 2.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption of the high- and mid-dose F0 males (12000 or 4000 ppm) was slightly but statistically significantly below control during premating weeks 0 - 1 (about 8 % or 6 %). Low-dose males (1500 ppm) did not show any test substance-related changes of food consumption.
Food consumption of the high-dose F0 females was statistically significantly below control between GD 14 - 20 (about 13 %) and between PND 1 - 4 (about 27 %). High-dose F0 females during premating and mid and low-dose females during all study phases did not show any test substance-related changes of food consumption.

HAEMATOLOGY
In females of all dose groups the absolute neutrophil counts (not statistically significant in dose group 1 (1500 ppm)) as well as the absolute monocyte counts were increased. The total white blood cell (WBC) counts in females of the mentioned dose groups were also higher compared to controls, although not statistically significant. The medians of the mentioned parameters were higher compared to the historical control ranges of 3 month old female Wistar rats (WBC: 2.85 – 4.21 G/L; absolute neutrophil counts 0.40 – 0.93 G/L; absolute monocyte counts 0.04 – 0.10 G/L).

CLINICAL CHEMISTRY
In females of dose groups 2 and 3 (4000 and 12000 ppm) the albumin levels were decreased.
In female and male rats of dose groups 2 and 3 (4000 and 12000 ppm) and additionally in females of dose group 1 (1500 ppm) the alanine aminotransferase (ALT) activities were statistically significantly increased. However, in males of dose group 2 (4000 ppm) and in females of dose group 1 and 2 (1500 and 4000 ppm) the ALT values were within the historical control ranges (ALT males: 0.71 – 0.94 μkat/L; females: 0.42 – 0.73 μkat/L) and therefore were regarded as maybe treatment-related but not adverse. In rats of both sexes of dose group 3 (12000 ppm) and additionally in females of dose group 2 (4000 ppm) the calcium levels were statistically significantly increased but all values were within the historical control ranges (calcium males: 2.52 – 2.76 mmol/L; females 2.53 – 2.77 mmol/L) and therefore were regarded as incidental and not treatment-related.

URINALYSIS
No treatment-related changes among urinalyses parameters were measured.

NEUROBEHAVIOUR
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observations and the open field observations.
There were no test substance-related findings in male and female animals of all test groups in sensorimotor tests. Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore, these observations were considered as being incidental.
No statistically significant changes on motor activity data (summation of all intervals) was observed in the male and female animals of all dose groups in comparison to the concurrent control group.

ORGAN WEIGHTS
Statistically significant weight differences recorded for the ovary, spleen, adrenal gland, liver and kidney were considered to be test item-related.
Most prominent organ weight changes measured in this study were in the ovaries. Corroborating the macroscopic and histopathological observations, mean absolute and relative ovary weights were statistically significantly higher in all treated female groups. The effect was dose-related. Mean absolute and relative spleen weights were statistically significantly higher in all treated female groups, the difference to controls being strongest in the group administered 4000 ppm. No effect was noted on spleen weights of the males. Mean absolute adrenal gland weights were higher in both sexes administered 4000 ppm, and mean relative adrenal gland weights were higher in both sexes administered 4000 or 12000 ppm. The difference to controls was minor in severity and lacked a dose relationship. Mean absolute and relative liver weights were statistically significantly higher in the female group administered 4000 ppm. In the females treated at 12000 ppm, only the relative liver weight was statistically significantly increased. The effect was minor in severity and was not seen in the males. Mean absolute kidney weights were statistically significantly higher in females administered 1500 or 4000 ppm, and mean relative kidney weights were statistically significantly higher in severity and not dose-related. There was no effect on kidney weights of the males. Some other statistically significant organ weight differences to controls were recorded in this study. Mean absolute and relative epididymis weights were minimally higher in males treated at 4000 ppm, but this variation was considered incidental in the view of its low magnitude and lack of dose relationship. Compared to controls, mean absolute heart weight was minimally lower and mean relative brain weight was minimally higher in the female group administered 12000 ppm. This difference was interpreted to be a consequence of the lower mean terminal body weight recorded in this group and thus not to be directly treatment-related. All other organ weight variations noted in this study were not statistically significant and considered to be fortuitous.

GROSS PATHOLOGY
At terminal necropsy, one female of the control group, one female treated at 1500 ppm, two females treated at 4000 ppm and one female treated at 12000 ppm were found not to be pregnant.
Macroscopic organ changes associated with histopathological lesions and therefore considered to be test item-related were seen in the females. Corresponding to organ weight changes, enlarged ovaries were seen in a dose-related manner, in females of all three treated groups. Spleen and/or mesenteric lymph node were enlarged in occasional females treated at 4000 or 12000 ppm. In single females of the high dose group, only, axillary lymph nodes, mediastinal lymph nodes or liver lymph node were enlarged. Also, prominent liver foci in one female administered 12000 ppm were interpreted to be test item-related in view of the corroborative histopathological changes seen.
Other macroscopic organ findings were very few and considered to be incidental and not treatment-related.

HISTOPATHOLOGY: NON-NEOPLASTIC
Small intestine:
In the small intestine, minimal villous infiltrates of histiocytes and/or mixed cells were seen in the jejunum, duodenum and/or ileum in a small proportion of animals treated at 12000 ppm and in single females treated at 1500 or 4000 ppm (jejunum only). In single females, these small intestinal changes were accompanied by minimal focal abscess(es)/necrotic foci and/or mural histiocytic/mixed cell infiltrates.
Peyer's patch:
In the small intestinal Peyer's patch, histiocytic/mixed cell infiltrates were seen in a dose related manner in some females treated at 4000 or 12000 ppm and in one male treated at 12000 ppm. The occurrence of minimal infiltrates in isolated females of the control group and low dose group was considered to be fortuitous and unrelated to the treatment.
Mesenteric lymph node:
Minimal to massive degrees of histiocytic/mixed cell infiltrates were also seen in the mesenteric lymph node (draining lymph node of the small intestine), in a dose-related manner in males and females of all dose groups, with a tendency of showing higher degrees of severity in the females. These changes were associated with minor lymphoid hyperplasia of the lymph node in a proportion of males and females of all dose groups, and with abscesses/necrotic foci in a proportion of females treated at 12000 ppm and one single female treated at 4000 ppm. Moreover, in a proportion of treated females of all dose groups, mixed cell infiltrates were seen in the tissue adjacent to the lymph node.
Stomach:
Minimal or slight degrees of a focally extensive submucosal edema/inflammation were observed in a proportion of males and females treated at 4000 or 12000 ppm and were seen mostly at the glandular portion of the stomach.
Liver:
The liver of one female treated at 12000 ppm showed a moderate degree of histiocytic/mixed cell infiltrates with necrosis, confirming the macroscopic observation of liver foci in this animal. This change was considered test item-related. No histopathological change was found to corroborate the liver weight differences recorded at necropsy.
Adrenal gland:
In the adrenal gland, mixed cell infiltration and single cell death in the zona fasciculata were seen in one female each of the 4000 ppm and 12000 ppm dose group and might be related to test item administration. Moreover, diffuse cortical hypertrophy was noted in some females administered 4000 or 12000 ppm. This minor change might account for adrenal gland weight changes noted at necropsy and was considered to possibly represent
a metabolic adaptation of the adrenal cortex to treatment.
Spleen:
In the spleen, a minimal mixed cell infiltration was seen in two females treated at 12000 ppm. This was probably a consequence of the inflammatory small intestinal and lymph node changes and therefore considered test item-related.
Bone marrow:
Likewise, in the femoral bone marrow, a minimally or slightly increased myeloid:erythroid ratio was noted in a proportion of females treated at 4000 or 12000 ppm and was considered to represent increased granulopoiesis as a consequence of the inflammatory state in these animals.
Other organs:
In single females administered 12000 ppm, the axillary, mediastinal and/or liver lymph node showed histiocytic/mixed cell infiltrates with or without presence of abscess(es)/necrotic foci, and corresponding to changes noted in the mesenteric lymph node. These lesions were therefore considered test item-related. The urinary bladder of a single female treated at 12000 ppm showed minimal mural histiocytic/mixed cell infiltrates, which were also considered test item-related. In the kidney, no histopathological changes were found to account for the higher kidney weights noted in the test item-treated groups.
A number of other histopathological findings were noted in treated and/or control rats, but were considered to be most likely incidental and/or to be within the range of expected changes for Crl:WI(Han) rats of this age kept under experimental conditions.

REPRODUCTIVE ORGANS
Ovary:
In the corpora lutea of the ovary, a foamy change with mixed cell inflammation was observed in all test item-treated females, the mean severity of the change being clearly dose-related.

No other histopathological lesion considered to be test item-related was noted in the male or female reproductive organs.
At terminal necropsy, one control female and four test item-treated females were found not to be pregnant. However, no dose relationship was observed for this finding, and no relevant histopathological findings were seen in mates of the females concerned. Therefore, occasional failure of pregnancy could not be attributed to the treatment in the present study.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

DISCUSSION

Tris-(2-ethylhexyl)amin O 2446 was administered as a constant homogeneous addition to the food in different concentrations (1500, 4000 and 12000 ppm) to groups of 10 male and 10 female Wistar rats (F0 animals). These concentrations in feed correspond to about 105, 277 and 815 mg/kg bw/d in males; 103, 271, 770 mg/kg bw/d in non-pregnant females; 121, 306 and 918 mg/kg bw/d in pregnant females; 173, 455, 1098 mg/kg bw/d in lactating females.

 

Clinically, toxicity was noted in the F0 males and F0 females at 4000 and 12000 ppm. Salient clinical findings were decreases in food consumption and body weights/ body weight gain which were most severe in females during gestation and lactation and led to a minimally lower mean terminal body weight in females administered 12000 ppm.

Detailed clinical examinations in an open field, detailed observations in a functional observational battery (FOB) and measurements of motor activity did not reveal any indications of test substance-induced effects in low-, mid- and high-dose rats (1500 ppm [125 mg/kg bw/d], 4000 ppm [327 mg/kg bw/d] and 12000 ppm [900 mg/kg bw/d]).

Clinical pathology revealed that in all dosed female rats the absolute neutrophil and monocyte counts and correspondingly the total white blood cell counts were elevated. This was most probably related to inflammatory changes in various organs.

The increased serum ALT activities in female and male rats of the high dose group (12000 ppm) indicated a slight degradation of the liver cell membranes. The lower albumin levels in females of dose group 2 and 3 (4000 and 12000 ppm) could be interpreted as an altered liver cell metabolism in these animals.

Pathology and histopathology revealed a multitude of test substance-related findings. Treatment led to minimally lower mean terminal body weight in females administered 12000 ppm and to prominent, dose-related ovary weight increases in all treated female groups. Other minor organ weight increases considered to be test substance-related were observed for the spleen, adrenal gland, liver and kidney, predominantly in the females, but were not clearly dose-related.

Test substance-related macroscopic organ changes were seen in the females, only, and mainly comprised enlarged ovaries in all dose groups and enlarged spleen and/or mesenteric lymph node in occasional females treated at 4000 or 12000 ppm.

Predominant histopathological findings were dose-related histiocytic/mixed cell infiltrates in multiple organs, with an overall tendency of being more severe in the females.

In the small intestine, minimal villous infiltrates of histiocytes and/or mixed cells were seen in some males and females treated at 12000 ppm and in single females of the lower dose groups. In individual females, these small intestinal changes were accompanied by minimal focal abscess(es)/necrotic foci and/or mural histiocytic/mixed cell infiltrates. Histiocytic/ mixed cell infiltrates were also seen in the Peyer's patch in a dose-related manner in some females treated at 4000 or 12000 ppm and in one male treated at 12000 ppm. Moreover, dose-related minimal to massive degrees of histiocytic/mixed cell infiltrates were noted in the mesenteric lymph node (draining lymph node of the small intestine) in the majority of males and females of all dose groups, in some animals together with lymphoid hyperplasia, abscesses/necrotic foci and/or adjacent mixed cell infiltrates. The axillary, mediastinal and/or liver lymph node of single females administered 12000 ppm showed similar findings.

In the stomach, minimal or slight degrees of a focally extensive submucosal edema/ inflammation were observed in a proportion of males and females treated at 4000 or 12000 ppm. The urinary bladder of a single female treated at 12000 ppm showed minimal mural histiocytic/mixed cell infiltrates, which were also considered test substance-related.

In the corpora lutea of the ovary, a prominent foamy change with mixed cell inflammation was observed in all test substance-treated females, the mean severity of the change being clearly dose-related.

The liver of one female treated at 12000 ppm showed a moderate degree of histiocytic/mixed cell infiltrates with necrosis, which was considered test substance-related. No histopathological change was found to corroborate the liver weight differences recorded at necropsy.

In the adrenal gland, mixed cell infiltration and single cell death in the zona fasciculata were seen in one female each of the 4000 ppm and 12000 ppm dose group and might be related to test substance administration. Moreover, diffuse cortical hypertrophy was noted in some females administered 4000 or 12000 ppm. This minor change might account for adrenal gland weight changes noted at necropsy and was considered to possibly represent a metabolic adaptation of the adrenal cortex to treatment.

It can be speculated that the infiltrates in the small intestine, draining lymph nodes and other organs might represent histiocytes containing test substance-lipid complexes which are poorly degraded by lysosomal enzymes and lead to a secondary local inflammatory reaction. Ovarian changes might also represent storage of test substance-lipid complexes with inflammation and have been described in the literature to occur after administration of amphophilic cationic chemical entities.

Minimal mixed cell infiltration in the spleen of two females treated at 12000 ppm, as well as increased granulopoiesis in the bone marrow of female dose groups treated at 4000 or 12000 ppm were probably a consequence of the inflammatory small intestinal and lymph node changes.

 

 

CONCLUSION

No NOAEL for general, systemic toxicity of the test substance was determined for the F0 parental animals based on clinical-pathological and histopathological findings in all treated groups, predominantly dose-related histiocytic/mixed cell infiltrates in multiple organs (ovaries, intestines and their draining lymph nodes) which lead to secondary local inflammatory reactions. These inflammations, above all in the ovaries, presumably also caused a slight adverse effect on reproductive performance as indicated by slightly lower numbers of implants in all treated groups. Females were more sensitive than males.

Applicant's summary and conclusion