Registration Dossier

Administrative data

Description of key information

oral

For the oral route, several studies are available. The most import and most reliable studies are an OECD TG 408 and an OECD TG 443, which are used in an weight of evidence approach:

In the 90-day (gavage) study with rats (OECD TG 408 and GLP) dose levels of 2.5, 12 and 60 mg/kg bw/day were administered resulting in a NOAEL of 2.5 mg/kg bw/day. The 3-month administration of the test item to male and female rats at a dose of 60 mg/kg bw/day each working day by gavage led to clear toxic findings, such as reduced feed consumption, retarded body weight gain, impaired general state of health, changes of the hematologic (white and red blood counts), enzymatic, clinicochemical and urinanalytical parameters as well as hepato-and nephrotoxic, myocardially toxic and adrenotoxic (progressive transformation) effects. A dose of 12 mg/kg bw/day led in the female animals to a reduced feed consumption, in the animals of both sexes to a retarded body weight gain, and to an increase of the aspartate aminotransferase values in the male animals. Urinalysis detected in both sexes an increased number of bacteria, of round-cell epithelia with and without nucleus, and of renal epithelia and in the male animals an increase of erythrocytes. The pathological examinations exhibited hepato and nephrotoxic and myocardially toxic findings. The dose group of 2.5 mg/kg bw/day caused no differences when compared with the control and was defined as NOAEL. [BASF AG, 1990]

Furthermore, DMDC was administered in an EOGRTS (OECD TG 443 and GLP) to groups of 25 male and 25 female healthy young Wistar rats as test groups 00 - 03 as an aqueous preparation by stomach tube at different dosages (0, 1.5, 5 and 15 mg/kg body weight/day [mg/kg bw/d]). F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Control animals were dosed daily with the vehicle (0.5% Sodium carboxymethyl cellulose [CMC] suspension in drinking water). Under the conditions of the present extended 1-generation reproduction toxicity study the NOAEL for general, systemic toxicity is the low dose of 1.5 mg/kg bw/d for the F0 animals. This was based on treatment-related, adverse effects such as a reduction in water and food consumption, decrease in body weight (change), altered clinical pathology parameters as well as histopathological changes in several organs, which were observed at the high- and mid-dose of 15 and 5 mg/kg bw/d. [BASF SE, 2020].

Inhalation

For the inhalation route, a 90-day study with rats (OECD TG 413 and GLP) was performed with concentrations of 2, 12 and 48 mg/m3. The NOAEC was 12 mg/m3 for local and systemic effects. Repeated inhalation of the test item showed systemic toxicity in the target organs liver and spleen in the form of hepatotoxicity and a disturbed hemoglobin metabolism. The amine-specific local toxicity was manifest in the form of changes in the target organs skin and nasal mucosa and led to hyperkeratosis or to a degeneration of the olfactory epithelium. [BASF AG, 1992]

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24th February 1987 - 12th September 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr. Karl Thomae GmbH, Biberach, Germany
- Age at study initiation: 42 days
- Weight at study initiation: males: 178 (168 - 193) g; females: 147 ( 139 - 157) g
- Housing: single
- Diet: Kliba-Labordiaet Ratte/Maus/Hamster Haltung GLP 343 Mehl, ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 % CMC (carboxymethyl cellulose)
Analytical verification of doses or concentrations:
yes
Remarks:
gas chromatography
Details on analytical verification of doses or concentrations:
Stability and homogeneity of the test item preparation in 0.5% CMC was confirmed.
Duration of treatment / exposure:
3 month
Frequency of treatment:
each working day (5 days/week)
Dose / conc.:
2.5 mg/kg bw/day (actual dose received)
Dose / conc.:
12 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before and after the application period
- Dose groups that were examined: control and high dose

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 29 and 85
- Anaesthetic used for blood collection: No data
- Animals fasted: yes, overnight
- How many animals: all
- Parameters examined: Erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), thrombocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 29 and 85
- Animals fasted: Yes
- How many animals: all
- Parameters examined: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyltranspeptidase, sodium, potassium, chloride, inorganic phosphorus, calcium, bilirubin total, creatinine, glucose, urea, total protein, albumin, cholesterol, triglycerides.

URINALYSIS: Yes
- Time schedule for collection of urine: day 23 and 79
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: Nitrite, pH-Value, hemoglobin, protein, glucose, ketone bodies, bilirubin, urobilinogen, sediment, volume.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
blood was sampled from all surviving animals of both sexes for immunological determinations after about 8 and 13 test weeks.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
DUNNETT-test
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
60 mg/kg bw/day: Deteriorated general state of health with differently discolored regions of various localizations in the animals of both sexes was observed.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
60 mg/kg bw/day: On day 71 one male was sacrificed moribund.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
60 mg/kg bw/day: Significantly retarded body weight gain in both sexes.
12 mg/kg bw/day: Significantly retarded body weight gain in the female animals, which was present in the males only as a trend.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
60 mg/kg bw/day: Reduced feed consumption in the males and females.
12 mg/kg bw/day: Slight reduction of the feed consumption in the females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
60 mg/kg bw/day: Increase of the lymphocyte values with changed nuclear structure in both sexes. Increase of the monocyte and neutrophilic polymorphonuclear granulocytes in the females. Decrease of the mean cell volume, mean hemoglobulin content of the individual erythrocyte, of the chloride and creatinine values in both sexes. Decrease of the total protein, albumin, globulins and triglyceride levels in the males. Increased inorganic phosphate in the females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
60 mg/kg bw/day: Increase of the alanine aminotransferase and aspartate aminotransferase in both sexes.
12 mg/kg bw/day: Increase of the aspartate aminotransferase values in the males.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
60 mg/kg bw/day: Increase of the erythrocyte and leukocyte values, of renal and round-cell epithelia, bacteria and roundcell epithelia without nucleus in the urine of both sexes.
12 mg/kg bw/day: Increase of bacterias and round-cell epithelias without nucleus in the urine of both sexes. Increase of erythrocytes in the urine of the males and single renal and round-cell epithelias in both sexes
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Description (incidence and severity):
The immunological examinations elicited no adverse effects on the humoral parameters examined.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
60 mg/kg /bw/day: Increase of the absolute liver weights in the females. Increase of the relative liver weights in the males and females. Increase of the relative kidney weights of the males and females. Increase of the absolute adrenal weights of the males and females. Increase of the relative adrenal weights of the males and females. Decrease in absolute (-18.6%) and an increase in relative (+40.5%) testis weight in the high doses males was detected as a consequence of the reduced body weight and cachexia.
12 mg/kg bw/day: Increase of the relative liver weights in the males. Increase of the absolute kidney weights in the males. Increase of the relative kidney weights in the males and females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
60 mg/kg bw/day: In all males of this dose group smaller seminal vesicles and atrophy in testis were observed as a consequence of the reduced body weight and cachexia.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
60 mg/kg bw/day: Histopathology revealed microvacuolar degeneration of the liver of most animals. The lesion was qualitatively more distinct in the female than in the male animals. Vacuolar tubulopathy was seen in the kidneys of all animals that were sacrificed at the end of the study. Vacuolar myocardial degeneration was observed in the heart of all male and female animals. The adrenal glands of all male and female animals showed the picture of a progressive transformation. Atrophy of the seminiferous tubuli (4/10 focal, 2/10 diffuse) and reduced contents of the seminal vesicles (10/10) in high dose males was noted.
12 mg/kg bw/day: Histopathology revealed vacuolar tubulopathy in the kidneys of some male and female animals. The heart of most animals was found to show vacuolar myocardial degeneration
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
2.5 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
12 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
60 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
60 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
blood
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
12 mg/kg bw/day (actual dose received)
System:
cardiovascular
Organ:
heart
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
60 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
adrenal glands
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD 443
Version / remarks:
25 June 2018
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Name of test substance: 2,2’-dimethyl-4,4’-methylenebis(cyclohexylamine)
Test substance No.: 00/0695-4
Batch identification: 69518616K0
Purity: 100 area-% (complex mixture of isomers)
Identity: Confirmed
Homogeneity: Given
Storage stability: Expiry date: 15 Jul 2019
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is the preferred animal species for developmental and reproductive toxicity studies according to the various test guidelines. This Wistar rat strain (Crl:WI(Han)) is selected because extensive historical control data is available for these rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Charles River Laboratories, Research Models and Services, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 5 weeks
- Housing:
Polysulfonate cages Typ 2000P
Exceptions:
From delivery to randomization (F0 animals), during mating, gestation, lactation, females after weaning, for functional observational battery and motor activity measurements: Polycarbonate cages type III
Polycarbonate cages : 1 animal (Exceptions during mating: 1 male/1 female per cage and during rearing up to PND 21/22: 1 dam with her litter)
- Diet: Ground Kliba maintenance diet mouse/rat “GLP”, Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: Drinking water ad libitum
- Acclimation period:
7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
20-24
- Humidity (%):
45-65
- Air changes (per hr):
15
- Photoperiod (hrs dark / hrs light):
12/12
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% CMC suspension in drinking water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the test substance preparation, the specific amount of test substance will be weighed, topped up with 0.5% Sodium carboxymethyl cellulose (CMC) suspension in drinking water in a calibrated beaker and intensely mixed with a magnetic stirrer.
Before and during administration, the preparations will be kept homogeneous with a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: The test item is soluble in 0.5% CMC suspension in drinking water.
- Concentration in vehicle: 0.015 g/100 mL, 0.05 g/100 mL, 0.15 g/100 mL
- Amount of vehicle: 10 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in 0.5% CMC suspension in drinking water at room temperature over a period of 7 days had been verified prior to the start of the study in a similar batch.
Duration of treatment / exposure:
The F0 animals, with the exception of the controls, will receive the test substance daily by gavage according to the time schedule (exception: no administration to animals being in labor) for approximately 10 weeks prior to breeding and continuing through breeding (up to two weeks), and for a maximum of 6 post-mating weeks (males) or gestation (three weeks) and lactation (three weeks) for females.
Frequency of treatment:
Once daily
Dose / conc.:
1.5 mg/kg bw/day (nominal)
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
No. of animals per sex per dose:
F0: 25/sex/group
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the administration period (only F0 parents) on day 0 and subsequently once per week (as a rule in the morning)
- Parameters observed:
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 (Protruding eyeball)
15. Assessment of the feces excreted during the examination (appearance/consistency)
16. Assessment of the urine excreted during the examination
17. Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: In general, the body weight of the male and female F0 parental animals, F1 rearing animals was determined once a week at the same time of the day (in the morning). The body weight of the F1 rearing animals was determined on the first day of test substance administration and then once a week at the same time of the day (in the morning).
The following exceptions are notable for the female parental animals:
• During the mating period of the F0 parental animals, the 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 of parturition (PND 0) and on PND 1, 4, 7, 10, 14, 18 and 21.
Females without positive evidence of sperm, females without litter and females after weaning (PND 21/22) were weighed once a week together with the males

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Generally, food consumption was determined once a week (over a period of 7 days) for the male and female F0 and F1 rearing animals, with the following exceptions:
• Food consumption will not be determined after the 10th 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 GD 0-7, 7-14 and 14-20.
• Food consumption of the F0 females, which gave birth to a litter, was determined for PND 1-4, 4-7, 7-10, 10-14, 14-18 and 18-21.
Food consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Generally, water consumption was determined once a week (over a period of 3 days) for the male and female F0 and F1 rearing animals, with the following exceptions:
• Water consumption was not determined after the 10th premating week (male F0 animals) and during the mating period (male and female F0 parental animals).
• Water consumption of the F0 females with evidence of sperm was determined for GD 0-1, 3-4, 7-8, 10-11, 14-15, 17-18 and 19-20.
• Water consumption of the F0 females, which gave birth to a litter, was determined for PND 1-2, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21.
Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.
Sacrifice and pathology:
SACRIFICE
All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS (F0)
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands (fixed)
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Lymph nodes, axillary
10. Lymph nodes, mesenteric
11. Ovaries
12. Pituitary gland (fixed)
13. Prostate (ventral and dorsolateral part together, fixed)
14. Testes
15. Seminal vesicles including coagulating gland (fixed)
16. Spleen
17. Thymus (fixed)
18. Thyroid glands (with parathyroid glands) (fixed)
19. Uterus (with cervix)
All paired organs were weighted together (left and right).

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. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix uteri
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left (fixed in modified Davidson´s solution)
11. Esophagus
12. Eyes with optic nerve (fixed in modified Davidson’s solution)
13. Heart
14. Ileum
15. Jejunum (with Peyer’s patches)
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries (fixed in modified Davidson´s solution)
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Rectum
28. Sciatic nerve
29. Seminal vesicles
30. Skeletal muscle
31. Spinal cord (cervical, thoracic and lumbar cord)
32. Spleen
33. Stomach (forestomach and glandular stomach)
34. Target organs
35. Testis, left (fixed in modified Davidson ´s solution)
36. Thymus
37. Thyroid glands (with parathyroid glands)
38. Trachea
39. Urinary bladder
40. Uterus
41. Vagina
42. Vas deferens

The left testis and left epididymis of all male F0 parental animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters.
In case of macroscopic findings in the right testis or right epididymis, this testis as well as the corresponding epididymis were fixed for histopathological examination and the left testis and epididymis were used for sperm analysis.
For technical reasons, after about 24 hours fixation the ovaries of F0 females of all test groups will be transferred to 70% ethanol.
The uteri of all cohabited female F0 parental animals will be examined for the presence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method (1)). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCl).
Spleens of 10 animals per sex per group of cohort 1A were split in two comparable parts (transversally). One part of the spleen was fixed in 4% buffered formaldehyde and afterwards was embedded in paraplast. The other part of the spleen was frozen at -80°C, being used to perform a splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells).

Reproductive organs of all F0 parental animals suspected of reduced fertility, or for which estrous cyclicity or sperm quality were affected, have been subjected to histopathological investigation. Organs demonstrating potential treatment–related changes were examined in the lower dose groups. A differential ovarian follicle count (DOFC) was conducted in test groups 10 and 13 (cohort 1A females) according to Plowchalk et.al.

CLINICAL PATHOLOGY
Clinical Pathology in F0 parental animals
Samples were withdrawn from the first 10 surviving F0 parental (females with litter, corresponding males) males and females per group at termination.
Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia.
In the afternoon preceding the day of urinalysis, the animals ere individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens were examined in a randomized sequence (the list of randomization instructions was compiled with a computer).
The following parameters were examined in all animals:
Hematology:
1. Leukocytes
2. Erythrocytes
3. Hemoglobin
4. Hematocrit
5. Mean corpuscular volume (MCV)
6. Mean corpuscular hemoglobin (MCH)
7. Mean corpuscular hemoglobin concentration (MCHC)
8. Platelets
9. Differential blood count
10. Reticulocytes
11. Blood smear (only evaluated preparations will be archived)
12. Prothrombin time

Clinical chemistry:
1. Alanine aminotransferase
2. Aspartate aminotransferase
3. Alkaline phosphatase
4. Serum γ-glutamyl transferase
5. Sodium
6. Potassium
7. Chloride
8. Inorg. phosphate
9. Calcium
10. Urea
11. Creatinine
12. Glucose
13. Total bilirubin
14. Total protein
15. Albumin
16. Globulins
17. Triglycerides
18. Cholesterol

Hormone evaluations:
1. T4 (thyroxine)
2. TSH

Urinalysis
1. Volume
2. Color
3. Turbidity
4. pH value
5. Protein
6. Glucose
7. Ketones
8. Urobilinogen
9. Bilirubin
10. Blood
11. Specific gravity
12. Microscopy of sediment

Statistics:
see table 1
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical observations for males and females (except gestation and lactation period)
In the high-dose group (15 mg/kg bw/d), one single high-dose male (No. 94) showed piloerection during study week 3 and transient salivation during study week 4 after the premating period. It is likely, that these temporary findings were induced by a bad taste of the test substance or local affection of the upper digestive tract. They are, however, not considered to be adverse toxicologically relevant findings.
In the high-dose group, eight high-dose females (Nos. 178, 183, 184, 190, 193, 195, 196 and 199) showed piloerection during study week 7 after the premating period. Since the finding was only transient during a short time period, it was not assessed as treatment-related and adverse.
One mid-dose female (No. 158) had a palpable mass during study weeks 6 - 7 after the premating period which is assessed as incidental and not related to treatment with the test compound. Two sperm negative control females (Nos. 101 and 110) and two sperm negative females of the high-dose group (Nos. 177 and 186) did not deliver F1 pups. This observation was not considered to be associated with the test compound.

Clinical observations for females during gestation of F1 litters
No treatment-related, adverse findings were observed in any of the the test groups.
One mid-dose female (No. 158) had a palpable mass during GD 20 – 21 and one sperm positive females of the low-dose group (No. 139 - 5 mg/kg bw/d) did not deliver F1 pups. Since the findings were not related to dose, they were not considered to be treatment-related.

Clinical observations for females and offspring during lactation of F1 litters
No treatment-related, adverse findings were observed in any of the the test groups.
One mid-dose female (No. 158) had a palpable mass during the entire lactation period.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of the high-dose F0 males were statistically significantly below the concurrent control values on premating day 28 onwards till the end of the study (up to 22%).
Mean body weights were statistically significantly below the concurrent control values for the high-dose F0 females on premating day 28 onwards till the end of the study (up to 14%) and for the mid-dose F0 females during gestation (GD 0 and 20: up to 5%) and during lactation (PND 4 – 18: 7%).
Mean body weights were comparable to the concurrent control values in the mid-dose females during the premating period and in the low-dose males and females and mid-dose males during the entire study period. In males, body weight change was statistically significantly below the concurrent control values for the high-dose group during premating days 14 - 63, 0 - 63 (79%, 22%, respectively) and study weeks 0 - 2, 3 - 4 and 0 - 4 after the premating period (up to -2.2 g vs. 8.6 g in control).
For the mid-dose males, body weight change was decreased during premating days 21 - 28, 35 - 42 and study weeks 0 - 4 after the premating period (about 13%, 17% and 18%, respectively).
Low-dose males showed only a decrease during premating days 7 - 14 (about 11%) without relation to dose. Therefore, this was assessed as incidental. Body weight change was statistically significantly below the concurrent control values for the high-dose females during premating days 0 – 7, 28 – 35, 0 – 63, GD 7 – 20 and 0 - 20 (about 15%, 47%, 18%, 23% and 11%, respectively) and for the mid-dose females during GD 14 - 20 and PND 1 - 4 (about 13% and 48%, respectively).
Body weight change was comparable to the concurrent control values in the mid-dose females during the premating period and in the low-dose females during the entire study period.
The statistically significantly increased body weight change in the mid-dose females during PND 7 - 10 and in the high-dose females during PND 14 - 18 was considered to be spontaneous in nature and not treatment-related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose F0 males was statistically significantly below the concurrent control values during the premating days 21 - 42, 56 - 69 and 0 - 69 (up to 12%, 15% and 9%, respectively).
Food consumption was statistically significantly below the concurrent control values for the high-dose F0 females during premating days 28 - 35, 42 - 49; GD 7 - 20 and the entire lactation period (up to 9%, 11%, 13% and 19%, respectively).
Mid-dose females showed a reduction in food consumption during lactation only (PND 1 – 18: up to 14%).
Food consumption was comparable to the concurrent control values in the mid-dose females during the premating and gestation period and in the low-dose males and females and middose males during the entire study period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was affected in males and females of the F0 high- and mid-dose groups:
In F0 males, mean water consumption values of the high- and mid-dose groups were statistically significantly below the concurrent control values during premating days 21 - 66 (up to 17) and 28 - 59 (up to 12%), respectively. In F0 females, water consumption was statistically significantly below the concurrent control values for the high- and mid-dose groups during premating, gestation and laction. For the high-dose group, the reduction was up to 21% below control during premating (days 21-51, 63-66), 18% during gestation (GD 14 - 15, 19 – 20) and 23% during lactation (PND 1 - 2 and 7 – 11). Mid-dose females showed a reduction up to 18% below control during premating (days 28 - 52, 63 – 66), 16% during gestation (GD 14 - 15, 19 – 20) and 19% during lactation (PND 1 – 2).

Water consumption of the low-dose F0 males and females was comparable to the concurrent control values throughout the entire study.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in males and females of test group 03 (15 mg/kg bw/d) platelet counts were significantly increased. Additionally, in males of the mentioned test group absolute and relative eosinophil counts were significantly decreased, whereas in females of test group 03 absolute and relative monocyte counts were significantly increased. These changes were regarded as treatment-related and adverse.
In males of test groups 01, 02 and 03 (1.5, 5 and 15 mg/kg bw/d) red blood cell (RBC) counts were significantly decreased and in males of test groups 02 and 03 hemoglobin and hematocrit values were significantly lower compared to controls. However, all mentioned values were not dose-dependently changed, and they were within historical control ranges (F0 males, RBC 8.03-9.04 tera/L; hemoglobin 8.6-9.5 mmol/L; hematocrit 0.406-0.438 L/L). In males of test group 02 (5 mg/kg bw/d), absolute reticulocyte counts were significantly increased but the alteration was not dose dependent. Therefore, the mentioned changes in this paragraph were regarded as incidental and not treatment related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in males and females of test group 03 (15 mg/kg bw/d) aspartate aminotransferase (AST) activities and inorganic phosphate levels were significantly increased. Additionally, in males of test group 03 triglyceride values were significantly higher compared to controls. These changes were regarded as treatment-related and adverse.
The following significant changes were regarded as incidental and not treatment-related, because the values were within historical control ranges: increased AST activities in males of test group 02 (5 mg/kg bw/d); decreased creatinine and glucose values in males and females of test group 03 (15 mg/kg bw/d); increased alanine aminotransferase (ALT) activities and urea values in males of test group 03; decreased calcium values in males of test groups 01, 02 and 03 (1.5; 5 and 15 mg/kg bw/d); decreased total bilirubin values in females of test groups 02 and 03 (F0 males, AST 1.37-2.21 μkat/L; creatinine 23.4-34.8 μmol/L; glucose 5.19-6.98 mmol/L; ALT 0.56-0.89 μkat/L; urea 3.75-6.08 mmol/L; calcium 2.48-2.62 mmol/L; F0 females, creatinine 28.0-41.3 μmol/L; glucose 5.04-6.01 mmol/L; total bilirubin 1.18-2.71 μmol/L).
The following significant changes were regarded as incidental and not treatment-related, because they were not dose-dependent: decreased total protein, albumin and globulin values in males of test group 02 (5 mg/kg bw/d); decreased glucose values in females of test group 01 (1.5 mg/kg bw/d).
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In the urine sediment of males of test group 03, increased numbers of transitional epithelial cells were observed. This finding in combination with the histopathology findings in the kidneys is regarded as treatment-related and adverse.
At the end of the administration period, in male and female rats of test group 03 (15 mg/kg bw/d) and in males of test group 02 (5 mg/kg bw/d) urine pH values were significantly decreased. This change is probably treatment-related because of the excretion of acidic metabolites of the administered compound, but it is not regarded as adverse, per se.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In test group 03 (15 mg/kg bw/d), terminal body weight was significantly decreased in males (-22%) and females (-12%) below the historical control values and was regarded as treatment related. Consequently, significant absolute weight decreases and/or significant relative weight increases were seen in several organs, that were considered secondary to the decreased terminal body weights. Significantly, but secondarily decreased absolute organ weights in males were considered the brain, prostate, seminal vesicles, spleen and thymus, and in females the spleen. Secondarily and significantly increased relative organ weights in males were considered: brain, cauda epididymis, epididymides, heart, liver, spleen, testes and thyroid glands, and in females: adrenal glands, brain, heart, kidneys, liver and pituitary gland. Although the significant relative weight increases observed in the adrenal glands (+41%), kidneys (+31%) and pituitary gland (+42%) of males in test group 03 appeared too high to be directly associated with the decrease of terminal body weight, the absolute weights of all three were within historical control values. Nevertheless, it cannot be excluded, that also a direct effect is involved in the weight increase of these organs, especially a histopathological correlate was seen.
In test group 02 (5 mg/kg bw/d), the terminal body weight in males was significantly reduced (-6%), generating significantly and secondary decreased absolute weights of the prostate, and significantly but secondary relative increases of the epididymides, kidneys and pituitary gland. In females, although the terminal body weight, was not significantly reduced, the minimal significant relative weight increases in liver and kidneys were rather attributed to the body weight decrease than to a treatment-related effect. The significant absolute and relative weight increase of the ovaries occurred without a dose-dependency and was considered incidental.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All 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.
Fertility:
The female animals (Nos. 101, 110, 139, 177 and 186) that were not pregnant and their male mating partners (Nos. 1, 39 and 77) did not show any macroscopic findings, whereas the male mating partners (Nos. 10 and 86) exhibited testes and epididymides of reduced size. Furthermore, the prostate and seminal vesicles were found reduced in size in animal No. 86.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in following organs: adrenal cortex, brain, esophagus, eyes with optic nerve, glandular stomach, heart, kidneys, left epididymis, liver, lungs, axillary and mesenteric lymph nodes, pancreas, pituitary gland, seminal vesicles and skeletal muscle. The main finding in all these target organs was a “microvesicular” type of cytoplasmic vacuolation, characterized by the presence of single to multiple vacuoles (depending on the organ) ranging from very fine to small (no larger than the nuclei of the cell). If the microvesicular vacuolation was so fine that vacuoles could not be distinguished individually within the cytoplasm, additional terms referred here as “ground glass” (finest vacuoles that cannot be individually differentiated), “foamy” or “granular” (vacuoles that can hardly be differentiated) were used for a better description. The cytoplasmic microvacuolation conferred the cells a very clear or transparent aspect and also increased their size. The finest type of vacuolation was frequently seen in epithelial cells. In the skeletal muscle, the vacuoles could be always individually visualized within the cells and had a particular birefringent aspect. Large cytoplasmic vacuoles, larger than the cell nuclei and often displacing them, were referred here as “macrovesicular” type of vacuolation and were only observed in the brain (choroid plexus) and seminal vesicles.

Adrenal cortex
In male animals of test group 03, the vacuolation of the zona fasciculata showed an increase in the incidence and grading. The vacuolation was characterized by the presence of a microvesicular pattern, giving the cytoplasm of the cells a foamy and pale aspect and often causing an increased size of the cells. This effect was clearly seen in males but not in females.

Axillary lymph node
The vacuolation of the high endothelial venules (HEV) was characterized by small vacuoles in the whole cytoplasm, which gave these vessels a conspicuous transparent aspect. Males were affected from test group 02 onwards and females only in test group 03.

Brain
The vacuolation was noted in the epithelial cells of the choroid plexus and was most frequently of microvesicular type. However, with increasing grading, macrovesicular vacuoles were also observed. A very tiny content was visible in these vacuoles, most likely representing membrane residues resulting from coalescence of smaller vacuoles. The choroid plexus of the lateral and dorsal third ventricles was affected most frequently. Only males and females in test group 03 were affected, with males showing a higher grading than females.

Esophagus
In the esophagus, the vacuolation was noted in the skeletal muscle layers of the wall. This finding was characterized by the presence of very tiny, transparent to birefringent microvacuoles ranging approximately from 2 – 4 μm in diameter within the muscle fibers. Males and females of test groups 02 and 03 appeared to be equally affected.

Eyes with optic nerve
A very fine microvesicular vacuolation was seen in the retinal pigment epithelium. The affected cells had ground-glass aspect and were minimally increased in size. This change was more manifested in the peripheral areas. Only animals in test group 03 were affected, with males having a slightly higher incidence and grading than females.

Glandular stomach
A microvesicular vacuolation (small vacuoles) was observed at the base of the glands of pyloric mucosa. Within the glandular cells, the vacuolation randomly displaced the nuclei from their basal position, giving the base of the glands a disorganized and paler aspect than normal. A dose-dependent increase from test group 02 onwards was noted in males and females.

Heart
The microvesicular vacuolation affected mainly the septum and left ventricular wall. It was characterized by multiple individual small vacuoles visible within the cardiomyocytes, without altering their shape or size. This change was not associated with visible degeneration or necrosis but conferred the cells a pale and disorganized aspect. Males appeared more affected than females.

Kidneys
In the kidneys the vacuolation was observed in the medulla (tubules of the inner stripe of the outer medulla) of both sexes, whereas the degeneration and/or regeneration was found in the cortex (proximal convoluted tubules) of males only. The vacuolation in the medulla was of a very fine microvesicular type, giving the epithelial cells a “ground glass” pale and swollen aspect. The tubular degeneration/regeneration was characterized by multifocal areas of convoluted tubules with microvesicular vacuolation, loss of normal architecture due to nuclear disorganization and crowding, single pyknosis and general light basophilia.

Left epididymis
The vacuolation was localized primarily in the ducts of approximately 2/3 of the distal corpus at the transition to the caudal region but did not include the cauda. The vacuolation of the epithelial cells ranged from small microvesicular vacuoles to vacuoles as large as the nuclei. The vacuoles were always located basal and lateral to the nuclei within the cytoplasm. A dosedependent increase in incidence and grading was noted from test group 02 onwards.

Liver
The vacuolation within the hepatocytes was characterized by very small cytoplasmic vacuoles of regular size distributed around the nuclei rather than in the periphery of the hepatocyte. This pattern was quite different from the “fatty change vacuolation” pattern, which is composed of vacuoles of different size. The mixed-cell inflammation observed particularly in females in test group 03, was composed predominantly of granulocytes and lymphocytes and was localized
in centrilobular areas affected by vacuolation. Often this type of inflammation was associated with apoptosis/single cell necrosis. Multinuclear hepatocytes in females were assumed to be the result of the coalescent damaged vacuolated hepatocytes. The vacuolation of the bile duct epithelium was of a “ground-glass” type affecting the whole cell and was observed in the portal bile ducts of large caliber. Their aspect was very pale, and the size of the epithelial cells was increased.

Lungs
The vacuolation was localized in the bronchial and bronchiolar epithelium and was characterized by a “foamy” aspect. In bronchi and large bronchioles, the vacuolation was mostly occupying the cytoplasm apical to the cell nuclei, whereas in the terminal bronchioles, the vacuolation was rather basal to the cell nuclei, displacing them to the apical region. In the bronchial associated lymphoid tissue (BALT), the high endothelial venules (HEV) showed the same type of vacuolation as described for the lymph nodes, with a noticeable pale aspect.

Mesenteric lymph node
Similarly, as observed in the axillary lymph nodes, the whole cells of the high endothelial venules (HEV) showed a microvesicular vacuolation (small vacuoles), conferring these vessels a very pale aspect. Macrophage aggregates were slightly increased in test groups 02 and 03 in males.

Pancreas
The cytoplasmic vacuolation of the acinar epithelium was of microvesicular. Within the acinar cells, very small vacuoles were localized in the apical cytoplasmic border adjacent to the zymogen granules (grade 1) or extended from the apical cell border to the cell nuclei (grade 2) accompanied by a reduction of zymogen granules. The ductal vacuolation was seen in the interlobular pancreatic ducts, with ground glass appearance of the epithelial cells accompanied by increase size.

Pituitary gland
The cytoplasmic vacuolation was localized in all cell types of the pars distalis. Within the cells the vacuolation was of a very fine microvesicular type involving the whole cytoplasm giving the cells a fine “granular” and pale aspect.

Seminal vesicles
The cytoplasmic vacuolation of the epithelial cells was characterized by small vacuoles of very regular size localized at the basal part of the cells displacing the nuclei to the apical border. The cells appeared wider and the nuclei lost their regular arrangement along the epithelium/basal membrane. Some animals showed single vacuoles of macrovesicular type. The vacuolation was dose-dependent from test group 02 onwards.

Skeletal muscle
As already described for the skeletal muscle of the esophageal wall, the vacuolation of the skeletal muscle was also characterized by the presence of very tiny intracytoplasmic microvacuoles (few μm in diameter) with a birefringent aspect and a multifocal distribution pattern within the muscle fiber. In addition, degeneration and /or regeneration of single myofibers was noted. The degenerating fibers revealed slightly altered staining features (basophilic or strong hyaline stain) and variable thickness (retracted or swollen aspect). Some of these degenerated fibers showed regeneration (reparative response) characterized by numerous central nuclei within the fibers. In test group 03 vacuolation was strongly associated with degeneration/regeneration, whereas in test group 02 only few animals showed vacuolation only.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered incidental or spontaneous in origin and without any relation to treatment.

Fertility
The female animals (Nos. 101, 110, 139, 177 and 186) that were not pregnant and the male mating partners (Nos. 1, 39 and 77) did not show relevant microscopic findings, whereas the male mating partners (Nos. 10 and 86) exhibited severe diffuse atrophy of the testes, and aspermia, debris and cribiform change in the epididymides. These findings were consistent with the macroscopic reduced size of the respective organs and explained the impaired fertility. No correlate was found for the reduced in size of the prostate and seminal vesicles found in animal No. 86. However, the seminal vesicles displayed a moderate microvesicular vacuolation.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormones
In F0 males of test group 03 (15 mg/kg bw/d) significantly decreased T4 values were observed. This change was neither accompanied by any alteration of the TSH values nor by any histopathological change of the thyroids. The T4 and TSH values were within the historical control range (F0 males, T4 44.65-78.17 nmol/L; TSH 4.41-9.80 μg/L). No significant change of T4 and TSH values was observed in F0 females of test group 03. Therefore, this isolated change of the T4 values in males of test group 03 was regarded as incidental and not treatment related.
Key result
Dose descriptor:
NOAEL
Effect level:
1.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
haematology
clinical biochemistry
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 mg/kg bw/day (nominal)
System:
other: The administration of DMDC led to a “microvesicular” type of cytoplasmic vacuolation. The vacuolation was not limited to one special target organ or system, but ditribued largely in the whole body.
Organ:
heart
kidney
lungs
lymph node
pancreas
pituitary gland
seminal vesicle
stomach
other: stomach, skeletal muscle, epididymidis
Treatment related:
yes
Dose response relationship:
yes

see attachment: 90R0695_00R050_results and tables

Conclusions:
Under the conditions of the present EOGRTS (extended 1-generation reproduction toxicity study) the NOAEL (no observed adverse effect level) for general, systemic toxicity is the low dose of 1.5 mg/kg bw/d for the F0 and F1 animals. This was based on treatment-related, adverse effects such as a reduction in water and food consumption, decrease in body weight (change), altered clinical pathology parameters as well as histopathological changes in several organs, which were observed at the high- and mid-dose of 15 and 5 mg/kg bw/d.
Executive summary:

2,2’-dimethyl-4,4’-methylenebis(cyclohexylamine) was administered to groups of 25 male and 25 female healthy young Wistar rats as test groups 00 - 03 as an aqueous preparation by stomach tube at different dosages (0, 1.5, 5 and 15 mg/kg body weight/day [mg/kg bw/d]): F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Control animals were dosed daily with the vehicle (0.5% Sodium carboxymethyl cellulose [CMC] suspension in drinking water).

In general, analyses confirmed the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle.

 

Regarding clinical examinations, F0 males and females of the high- and mid-dose groups (15 and 5 mg/kg bw/d) showed signs of systemic toxicity consisting of treatment-related, adverse changes such as a reduction in food and water consumption and a decrease in body weight (change), the latter was the most affected parameter in F0 animals.

 

In the F0 parental high-dose group, reduced water and food consumptions were observed in males (up to 17 and 15% below control, respectively) and females (up to 23 and 19% below control, respectively). In the mid-dose group, water consumption was reduced in males and females (up to 12 and 19% below control) whereas food consumption was reduced exclusively in females (up to 14% below control). Mean body weight was decreased in males and females of the high-dose (up to 22 and 14% below control) and females of the mid-dose group (up to 7% below control). Body weight change was decreased in both sexes of F0 high and mid-dose animals. Body weight decreases were 11 and 6% below controls in males and females of cohort 1A, 12 and 6% in males and females of cohort 1B. In almost all cases, body weight change was correspondingly decreased. The reduction in food and water consumption of high-dose F0 and mid-dose F0 animals in combination with the body weight decreases summed up to a treatment-related and adverse assessment. However, corresponding decreased terminal body weights of males and females were confirmed by necropsy (see below). Therefore, this combination was assessed as treatment-related adverse.

 

All other parameters were not altered. No treatment-related adverse findings were observed in the low-dose (1.5 mg/kg bw/d) F0 animals.

 

Regarding clinical pathology, in F0 rats of both sexes in test group 3 (15 mg/kg bw/d) stress was indicated by decreased absolute and relative eosinophil cells in males, but by increased absolute and relative monocyte counts in females. In the mentioned individuals no liver parameters were relevantly changed apart from higher triglyceride levels in F0 males, but aspartate aminotransferase (AST) activities and inorganic phosphate levels were increased. These altered parameters in combination with lower body weights of the rats were most probably due to a retarded growth affecting bones (inorganic phosphate, ALP) as well as skeletal muscle (AST).

 

F0 generation parental animals

Regarding pathology, terminal body weight was significantly decreased in males (-22%) and females (-12%) of test group 03. They were below the historical control values and were therefore considered treatment-related. Consequently, significant absolute weight decreases and/or significant relative weight increases were seen in several organs, that were considered secondary to the decreased terminal body weight.

Histopathology revealed treatment-related findings in following target organs of males and females: brain, esophagus, eyes with optic nerve, glandular stomach, heart, kidneys, liver, lungs, axillary and mesenteric lymph nodes, pancreas, pituitary gland, and skeletal muscle. Furthermore, the adrenal cortex, left epididymis and seminal vesicles were affected only in male animals.

The main finding in all these organs was a “microvesicular” type of cytoplasmic vacuolation, characterized by the presence of very few to multiple vacuoles, ranging from very fine to small vacuoles (no larger than the nuclei of the cell). Characteristically, if the cytoplasmic vacuolation was abundant, the cells were very clear or pale and increased in size. Vacuoles larger than the cell nuclei were referred here as “macrovesicular” type of vacuolation, which was observed in few organs (brain and seminal vesicles). In 13 out of 16 target organs the vacuolation occurred alone without additional signs of cytotoxicity. Only in 3 out of 16 target organs (kidneys, liver and skeletal muscle) the vacuolation was associated with signs of cytotoxicity (degeneration/regeneration, inflammation and apoptosis/single cell necrosis).

A summary of the histopathological treatment-related findings is given as follows:

In test group 03 (15 mg/kg bw/d), the adrenal cortex of males showed a microvesicular vacuolation of the zona fasciculata (minimal to moderate), giving the cytoplasm of the cells a foamy and pale aspect and often causing an increased size of the cells. This finding might have contributed to the significant relative weight increase of the adrenal glands (+41%). Females were not affected. In the left epididymis, the vacuolation was localized in the ducts of approximately 2/3 of the distal corpus and never included the cauda. The vacuolation (minimal to moderate) ranged from small microvesicular vacuoles to vacuoles as large as the nuclei. In the seminal vesicles, the vacuolation of the epithelial cells (minimal to moderate) was characterized by small vacuoles of very regular size localized at the basal part of the cells displacing the nuclei to the apical pole. Single vacuoles of macrovesicular type were also present in the most severe cases.

 

In following organs of test group 03, males and females were affected:

In the axillary and mesenteric lymph nodes, small vacuoles (minimal to slight) were seen in the cells of the high endothelial venules (HEV). In the brain, a microvesicular and macrovesicular vacuolation (minimal to moderate) was noted in the choroid plexus of the lateral and dorsal third ventricles. Males were more severely affected than females. In the esophagus, the vacuolation was seen in the skeletal muscle of the wall (minimal to slight), with similar characteristics as observed in the gastrocnemius muscle: very tiny, transparent to birefringent microvacuoles ranging approximately from 2 – 4μm in diameter within the fibers. A very fine vacuolation in the eyes (minimal to slight) was localized in the retinal pigment epithelium. The affected cells displayed a ground glass aspect and were minimally enlarged. In the glandular stomach, the vacuolation characterized by small vacuoles (minimal to slight) were observed at the base of the glands in the pyloric mucosa. The heart showed a microvesicular vacuolation affecting mainly the septum and left ventricular wall. This change was not associated with degeneration or necrosis but conferred the cells a damaged aspect. In the kidneys the vacuolation was observed in the medulla (tubules of the inner stripe of the outer medulla) in males and females, whereas only in males, degeneration/regeneration was noted in the cortex (proximal convoluted tubules) and was minimal to moderate. The vacuolation in the medulla was of a very fine microvesicular type (minimal to slight), giving the epithelial cells a “ground glass” pale and larger aspect. The more severe findings in the kidneys of males might have contributed to significantly increase the relative weight (+31%). The degeneration/regeneration was characterized by multifocal microvesicular vacuolation with loss of normal architecture due to nuclear disorganization and crowding, single pyknosis and general light basophilia. In the liver, the vacuolation in the hepatocytes was characterized by very small vacuoles of regular size (foamy aspect), which was more severe in females (minimal to moderate) than males (minimal to slight). An associated mixed-cell inflammation (minimal to moderate) and multinucleated hepatocytes were observed particularly in females and not in males. This type of inflammation was often associated with apoptosis/single cell necrosis (minimal to slight). Furthermore, the epithelium of the portal bile ducts of large caliber also showed a vacuolation with a ground-glass appearance in both sexes. In the lungs, the vacuolation (minimal to slight) was localized in the bronchial and bronchiolar epithelium and was characterized by a “foamy” aspect. In the bronchial associated lymphoid tissue (BALT), the high endothelial venules (HEV) showed the same type of vacuolation (minimal) as described in the lymph nodes. In the pancreas, a microvesicular vacuolation of the acinar epithelium was diffusely distributed along the organ (minimal to slight). Furthermore, a vacuolation was seen in the interlobular pancreatic ducts, presenting a ground glass appearance of the epithelial cells accompanied by increase size (minimal to slight). In the pituitary gland, the vacuolation was localized in the cells of the pars distalis. Within the cells the vacuolation was of a very fine microvesicular type, giving the cells a fine “granular” and pale aspect. It remains uncertain, if the vacuolation in the pituitary gland of females might have contributed to the significant relative weight increase of +41%. In the skeletal muscle (gastrocnemius muscle), as already described for the skeletal muscle of the esophageal wall, the vacuolation was characterized by very tiny microvacuoles with a birefringent aspect, ranging approximately from 2 – 4μm in diameter, with a multifocal distribution pattern within the muscle fiber. The vacuolation was minimal to slight in males and minimal in females. In addition, degeneration and /or regeneration of the myofibers was noted in males and females.

 

In test group 02 (5 mg/kg bw/d), the incidence and/or grading of the treatment-related vacuolation was generally lower and was not associated with additional signs of cytotoxicity Organs showing vacuolation in males only were: axillary and mesenteric lymph nodes, heart, left epididymis, pituitary gland and seminal vesicles. Organs with treatment-related vacuolation affecting both males and females were: esophagus, glandular stomach, kidneys, lungs, pancreas and skeletal muscle.

 

In test group 01 (1.5 mg/kg bw/d), no treatment-related findings were noted.

 

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered incidental or spontaneous in origin and without any relation to treatment.

 

The morphological pattern and abundant distribution of the vacuolation in 16 target organs suggests that this finding represents most likely a systemic phospholipidosis (Nonoyama and Fukada, 2008; Rudmann et al, 2004). In a previous study (BASF project No.: 85R0695/00R047) with the same test substance, special stains (PAS, Oil-Red-Oil and Sudan black) revealed that the vacuolation most likely had a phospholipidic nature. Since a definitive diagnosis of phospholipidosis should rely on transmission electron microscopy or chemical analysis, a conclusive diagnosis for the presence of phospholipidosis cannot be done in this study. However, based on the systemic distribution of the vacuolation affecting organs involved in multiple vital functions (brain, eyes, heart, etc.), the cytoplasmic vacuolation associated or not with cytotoxicity in in males and females of test groups 03 and 02 was assessed as treatment-related and adverse.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1.5 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
OECD TG 443
Organ:
heart
kidney
liver
other: skeletal muscle

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (OECD 413)
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr. KarL Thomae GmbH, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: males: 247 (226 - 266) g, females: 171 (159 - 181) g
- Housing: single
- Diet: SSNIFF R 10 mm Pellets, ad libitum
- Water: water ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
inhalation
Type of inhalation exposure:
nose/head only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: 3.5, 1.5, and 2.8 µm, respectively.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
gas chromatography
Duration of treatment / exposure:
3 months
Frequency of treatment:
6 hours each working day (5 days/week)
Remarks:
Doses / Concentrations:
0.002, 0.012, 0.048 mg/l (2, 12, 48 mg/m3)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2.1±0.58 µg/l, 12.4±12.63 µg/l, 48.2±10.48 µg/l
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Dose selection rationale: pre study
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before and at the end of the study
- Dose groups that were examined: control and high dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before first exposure and at day 87.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all
- Parameters examined: Erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), leucocyte count, thrombocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before first exposure and at day 87.
- Animals fasted: No data
- How many animals: all
- Parameters examined: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, sodium, potassium, chloride, inorganic phosphorus, calcium, bilirubin total, creatinine, glucose, urea, total protein, albumin, cholesterol, triglycerides.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes, adrenal gland, bone with marrow, brain, esophagus, eyes, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lungs, ovary, pancreas, pituitary gland, salivary gland, skin, spleen, stomach testis, thymus, thyroid gland, urinary bladder, and uterus, aorta, sternum, skeletal muscles, lacrimal gland, accessorius sex glands, mesenteric lymph nodes, all macroscopic alterations.
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no mortalities in the control and high dose groups. One female at 2 µg/l and one male at 12 µg/l died intercurrently after 37 and 48 exposures, respectively. Deaths were judged to be of spontaneous nature.

Scattered occurrence of observations throughout all test groups without relation to dose were noted. No specific substance-related effect noted.

BODY WEIGHT AND WEIGHT GAIN
Compared to control animals statistically reduced mean body weight gain (p<0.01) and reduced body weight from day 50 onwards (p<0.01) was seen in high dose male rats. Body weight was reduced by approx 14% compared to controls on day 85. In high dose females body weight change was significantly reduced (p<0.05) from day 71 onwards. Terminal body weight in females was reduced by 8% and statistically different from controls animals. No other statistically significant effect on body weight parameters were noted.

OPHTHALMOSCOPIC EXAMINATION
no changes in any of the dose groups noted.

HAEMATOLOGY
Significant (p<0.05) reductions in hemoglobin, hemoglobin per erythrocyte, and in mean corpuscular hemoglobin concentration (MCHC) were noted in the male high dose rats only. Polychromatosis was noted.
Clotting test: statistically significant clotting time increase was only seen in females but not in males. This effect was not considered to be treatment related.

CLINICAL CHEMISTRY
Animals at 12 µg/l: statistically significant, but marginal increase of alkaline phosphatase (5.658 µkat/l vs. 4.949 µkat/l in controls) and GPT (glutamate pyruvate transaminase; 1.043 µkat/l vs. 0.845 µkat/l in controls) in male rats. GOT (glutamate oxalo-acetate transaminase) was not changed in male rats. Increase of alkaline phosphatase was only seen in this test group. No other change was noted in male or female animals.

Animals at 48 µg/l: statistically significant increase of GOT and GPT (but not alkaline phosphatase) compared with controls in male rats, but not in females rats. Activity of GPT in serum was 1.081 µkat/l vs. 0.845 µkat/l in control animals (p<0.01). A significant (p<0.01) decrease of serum triglycerides in high dose males was considered to result from a decreased food consumption which was assumed because of the reduced body weight development in this group. This finding was therefore regarded to be a secondary effect.

ORGAN WEIGHTS
Relative organ weight of liver, lung, and kidney was significantly increased in high dose male and female animals on the 1% or 5% level of significance. Relative weight of adrenals (p<0.05) and testes (p<0.01), and absolute lung weight (1.41 g vs. 1.18 g in controls) were significantly increased only in high dose male rats.

HISTOPATHOLOGY: NON-NEOPLASTIC
No effects in low and medium dose animal groups.
Effects in high dose animals included: Local irritative effects on the skin and slight hyperkeratosis in 7/10 male rats. Minimal to slight vacuolization of the craniodorsal olfactory epithelium in both male (2/10) and female (1/10 animals) rats. Significantly increased incidence of slight tubulonephrosis was noted in male rats only (6/10 vs. 1/10 in male controls; 9/10 females vs. 7/10 controls), and extramedulary haematopoesis in spleen was noted only in female rats (9/10). Hemosiderin was noted in spleen of all high dose animals.
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
12 mg/m³ air
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
12 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
OECD TG 413
Organ:
liver
spleen

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (OECD 413)
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr. KarL Thomae GmbH, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: males: 247 (226 - 266) g, females: 171 (159 - 181) g
- Housing: single
- Diet: SSNIFF R 10 mm Pellets, ad libitum
- Water: water ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
inhalation
Type of inhalation exposure:
nose/head only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: 3.5, 1.5, and 2.8 µm, respectively.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
gas chromatography
Duration of treatment / exposure:
3 months
Frequency of treatment:
6 hours each working day (5 days/week)
Remarks:
Doses / Concentrations:
0.002, 0.012, 0.048 mg/l (2, 12, 48 mg/m3)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2.1±0.58 µg/l, 12.4±12.63 µg/l, 48.2±10.48 µg/l
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Dose selection rationale: pre study
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before and at the end of the study
- Dose groups that were examined: control and high dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before first exposure and at day 87.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all
- Parameters examined: Erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), leucocyte count, thrombocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before first exposure and at day 87.
- Animals fasted: No data
- How many animals: all
- Parameters examined: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, sodium, potassium, chloride, inorganic phosphorus, calcium, bilirubin total, creatinine, glucose, urea, total protein, albumin, cholesterol, triglycerides.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes, adrenal gland, bone with marrow, brain, esophagus, eyes, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lungs, ovary, pancreas, pituitary gland, salivary gland, skin, spleen, stomach testis, thymus, thyroid gland, urinary bladder, and uterus, aorta, sternum, skeletal muscles, lacrimal gland, accessorius sex glands, mesenteric lymph nodes, all macroscopic alterations.
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no mortalities in the control and high dose groups. One female at 2 µg/l and one male at 12 µg/l died intercurrently after 37 and 48 exposures, respectively. Deaths were judged to be of spontaneous nature.

Scattered occurrence of observations throughout all test groups without relation to dose were noted. No specific substance-related effect noted.

BODY WEIGHT AND WEIGHT GAIN
Compared to control animals statistically reduced mean body weight gain (p<0.01) and reduced body weight from day 50 onwards (p<0.01) was seen in high dose male rats. Body weight was reduced by approx 14% compared to controls on day 85. In high dose females body weight change was significantly reduced (p<0.05) from day 71 onwards. Terminal body weight in females was reduced by 8% and statistically different from controls animals. No other statistically significant effect on body weight parameters were noted.

OPHTHALMOSCOPIC EXAMINATION
no changes in any of the dose groups noted.

HAEMATOLOGY
Significant (p<0.05) reductions in hemoglobin, hemoglobin per erythrocyte, and in mean corpuscular hemoglobin concentration (MCHC) were noted in the male high dose rats only. Polychromatosis was noted.
Clotting test: statistically significant clotting time increase was only seen in females but not in males. This effect was not considered to be treatment related.

CLINICAL CHEMISTRY
Animals at 12 µg/l: statistically significant, but marginal increase of alkaline phosphatase (5.658 µkat/l vs. 4.949 µkat/l in controls) and GPT (glutamate pyruvate transaminase; 1.043 µkat/l vs. 0.845 µkat/l in controls) in male rats. GOT (glutamate oxalo-acetate transaminase) was not changed in male rats. Increase of alkaline phosphatase was only seen in this test group. No other change was noted in male or female animals.

Animals at 48 µg/l: statistically significant increase of GOT and GPT (but not alkaline phosphatase) compared with controls in male rats, but not in females rats. Activity of GPT in serum was 1.081 µkat/l vs. 0.845 µkat/l in control animals (p<0.01). A significant (p<0.01) decrease of serum triglycerides in high dose males was considered to result from a decreased food consumption which was assumed because of the reduced body weight development in this group. This finding was therefore regarded to be a secondary effect.

ORGAN WEIGHTS
Relative organ weight of liver, lung, and kidney was significantly increased in high dose male and female animals on the 1% or 5% level of significance. Relative weight of adrenals (p<0.05) and testes (p<0.01), and absolute lung weight (1.41 g vs. 1.18 g in controls) were significantly increased only in high dose male rats.

HISTOPATHOLOGY: NON-NEOPLASTIC
No effects in low and medium dose animal groups.
Effects in high dose animals included: Local irritative effects on the skin and slight hyperkeratosis in 7/10 male rats. Minimal to slight vacuolization of the craniodorsal olfactory epithelium in both male (2/10) and female (1/10 animals) rats. Significantly increased incidence of slight tubulonephrosis was noted in male rats only (6/10 vs. 1/10 in male controls; 9/10 females vs. 7/10 controls), and extramedulary haematopoesis in spleen was noted only in female rats (9/10). Hemosiderin was noted in spleen of all high dose animals.
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
12 mg/m³ air
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
12 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
OECD TG 413

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral

1. WoE study (OECD TG 408):

In a subchronic oral toxicity study (OECD TG 408 and GLP), rats were exposed to 0, 2.5, 12 and 60 mg/kg bw/day by gavage over 3 months (BASF AG, 1990b). For comparison, one group of animals (10 per sex) was used as control. The feed consumption and body weight of the rats were weekly determined. The state of health of the rats was checked each day, and when the animals were weighed, they were additionally inspected and thoroughly examined for clinical signs. During the administration period two clinicochemical and two hematological examinations as well as two urinalyses were carried out . In addition, blood was sampled from all surviving animals of both sexes for immunological determinations after about 8 and 13 test weeks.

Before the beginning of test substance administration and towards the end of the study, ophthalmological examinations were carried out in the animals of the control and of the 60 mg/kg bw groups. After the 3-month administration period, all surviving animals were assessed by gross pathology. Subsequently, a histopathological examination was carried out.

The following findings were obtained and assessed or discussed as substance-induced:

60 mg/kg bw group:

- Reduced feed consumption in the males and females

- Significantly retarded body weight gain in both sexes

- Deteriorated general state of health with differently discoloured body regions of various localizations in the animals of both sex

- Increase of the alanine aminotransferase, aspartate aminotransferase, leukocyte and lymphocyte values in both sexes

- Increase of the lymphocyte values with changed nuclear structure in both sexes

- Increase of the monocyte and neutrophilic polymorphonuclear granulocytes in the females

- Decrease of the mean cell volume, mean hemoglobulin content of the individual erythrocyte, of the chloride and creatinine values in both sexes

- Decrease of the total protein, albumin, globulins and triglyceride levels in the males

- Increased inorganic phosphate in the females

- Increase of the erythrocyte and leukocyte values, of renal and round-cell epithelia, bacteria and round-cell epithelia without nucleus in the urine of both sexes

- Increase of the absolute liver weights in the females

- Increase of the relative liver weights in the males and females

- Increase of the relative kidney weights of the males and females

- Increase of the absolute adrenal weights of the males and females

- Increase of the relative adrenal weights of the males and females

Histopathology revealed micro-vacuolar degeneration of the liver of most animals. The lesion was qualitatively more distinct in the female than in the male animals. Vacuolar tubulopathy was seen in the kidneys of all animals that were sacrificed at the end of the study. Vacuolar myocardial degeneration was observed in the heart of all male and female animals. The adrenal glands of all male and female animals showed the picture of a progressive transformation.

12 mg/kg bw group :

- Slight reduction of the feed consumption in the females

- Significantly retarded body weight gain in the female animals, which was present in the males only as a trend

- Increase of the aspartate aminotransferase values in the males

- Increase of bacteria and round-cell epithelia without nucleus in the urine of both sexes

- Increase of erythrocytes in the urine of the males and single renal and round-cell epithelia in both sexes

- Increase of the relative liver weights in the males

- Increase of the absolute kidney weights in the males

- Increase of the relative kidney weights in the males and females

Histopathology revealed vacuolar tubulopathy in the kidneys of some male and female animals. The heart of most animals was found to show vacuolar myocardial degeneration.

2.5 mg/kg bw group:

- No differences in comparison with the control in the animals of both sexes

In conclusion, it can be stated that a 3-month administration of the test item to male and female rats at a dose of 60 mg/kg bw/day each working day by gavage led to clear toxic findings, such as reduced feed consumption, retarded body weight gain, impaired general state of health, changes of the hematologic (white and red blood counts), enzymatic, clinicochemical and urine-analytical parameters as well as hepato-and nephrotoxic, myocardially toxic and adrenotoxic (progressive transformation) effects. A dose of 12 mg/kg bw/day led in the female animals to a reduced feed consumption, in the animals of both sexes to a retarded body weight gain, and to an increase of the aspartate aminotransferase values in the male animals. Urinalysis detected in both sexes an increased number of bacteria, of round-cell epithelia with and without nucleus, and of renal epithelia and in the male animals an increase of erythrocytes. The pathological examinations exhibited hepato and nephrotoxic and myocardially toxic findings. The dose group of 2.5 mg/kg bw/day caused no differences when compared with the control. A NOAEL was achieved at 2.5 mg/kg bw/day.

Discussion:

In this 90 day oral repeated dose study a decrease in absolute (-18.6%) and an increase in relative (+40.5%) testis weight in the high doses males (Wistar rats) was detected. Furthermore, atrophy of the seminiferous tubuli (4/10 focal, 2/10 diffuse) and reduced contents of the seminal vesicles (10/10) in high dose males was noted. In these animals a marked reduction in body weight was observed, which was ca. 42% lower at study termination when compared to the control group. Furthermore, very poor health condition (cachexia) of the males in this dose group and reduced feed consumption per day was detected. The histopathological finding of atrophy observed in testis confirms as morphological correlate the reduced absolute testis weights. Since the body weight was reduced more than the testis weight, the relative testis weight was increased (see table 1). At 12 and 2.5 mg/kg bw/day no effects on testis were observed and the bodyweight development was not significantly impaired.

In the 90-day inhalation study (see below, OECD TG 413, GLP) performed with the test item the relative weight of testis was significantly increased only in the high dose group males (48 mg/m3) but no atrophy of testis was detected. Furthermore, in the reproductive/developmental study (OECD TG 422, GLP) performed with the test item a decrease in absolute testis weight was detected which was considered to be secondary to the terminal body weight decrease in the high dose group (15 mg/kg bw/day). However, no atrophy of testis after oral administration was detected in this study and no treatment-related effect on reproductive performance and fertility was observed.

There are publications available which deal with the effects of feed restriction in rats from less severe (Chapin et al., 1993) to severe (Oishi et al., 1978) body weight reduction and the consequences on the reproductive system. Chapin and co-workers found that a reduction of up to 30% (70% remaining) of the control body weight in Sprague-Dawley rats did not lead to any effects on testis weights after a period of 17 weeks (Chapin et al., 1993). In contrast, Oishi and colleges were able to show that food restriction (body weight reduction of 45%, remaining 55%) already over four weeks in male Wistar rats had a significant impact on the absolute as well as relative testis weights (Oishi et al., 1978). The findings of both studies fit well, while simultaneously indicating a threshold of testicular degeneration at around 30% body weight loss.

However, based on the findings reported, the observations of the Oishi study are considered more relevant for the DMDC case. Firstly, the high dose animals treated with 60 mg DMDC/kg bw, which is far beyond the maximum tolerated dose (MTD), showed a body weight loss far beyond that used in the Chapin study (DMDC: 42% vs. Chapin study: 30%) and secondly Oishi and co-workers used exactly the same rat strain even equal aged as applied in the OECD TG 408 with DMDC. Briefly summarized: Oishi and colleagues were able to demonstrate that rats with a terminal body weight loss of around 45% (55% remaining; 320 g in control vs. 176 g in food restricted animals) compared to control animals already showed a reduction in absolute testis weights of 15% (3.22 g in control vs.2.73 g in food restricted animals) and a relative increase of 53% (1.01 in control vs. 1.55 in food restricted animals), which is totally in line with the findings obtained with DMDC (body weight loss: 42%; decrease of absolute testes weight of 18.6%). The effects on absolute and relative testes weight reduction and increase, respectively were more pronounced when the body weight loss was further increased.

Conclusion:

Therefore, the observed atrophy in testis and reduced contents of seminal vesicles were interpreted as consequence of the marked impairment on body weight development and poor general state of the animals rather than a direct effect of the test item. The findings were thus also not taken into account for classification and labelling.

References:

OECD SIDS 2001 , 2,2'-dimethyl-4,4'-methylenebis(cyclohexylamine), CAS No. 6864-37-5

Chapin R. E., Gulati D. K., Barnes L. H., and Teague J. L. (1993). The Effects on Feed Restriction on Reproductive Function in Sprague-Dawley Rats. Fundamental and Applied Toxicology, 20, 23-29.

Oishi S., Oishi H. and Hiraga K. (1978). The Effect of Food Restriction for 4 Weeks on Common Toxicity Parameters in Male Rats. Toxicology and Applied Pharmacology, 47, 15-22.

Table 1: Body and testis weight at study termination from OECD 408

Dose (mg/kg bw/day)

Absolute body weight (g)

Feed Consumption/animal/day (g)

Absolute testis weight (g)

Relative testis weight (mean in % of bw)

control

408

25.2

3.64

0.893

2.5

406

25.4

3.51

0.864

12

388

24.9

3.59

0.925

60

236**

(-42%)#

19.1

 

2.96*

(-18.6%)#

1.255**

(+40.5%)

* p < 0.05; ** p < 0.01

# weight reduction in % compared to control

2. WoE study (OECD TG 443):

DMDC was administered to groups of 25 male and 25 female healthy young Wistar rats as test groups 0 - 3 as an aqueous preparation by stomach tube at different dosages (0, 1.5, 5 and 15 mg/kg body weight/day [mg/kg bw/d]): F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Control animals were dosed daily with the vehicle (0.5% Sodium carboxymethyl cellulose [CMC] suspension in drinking water). 

Regarding clinical examinations, F0 males and females of the high- and mid-dose groups (15 and 5 mg/kg bw/d) showed signs of systemic toxicity consisting of treatment-related, adverse changes such as a reduction in food and water consumption and a decrease in body weight (change), the latter was the most affected parameter in F0 animals.

In the F0 parental high-dose group, reduced water and food consumptions were observed in males (up to 17 and 15% below control, respectively) and females (up to 23 and 19% below control, respectively). In the mid-dose group, water consumption was reduced in males and females (up to 12 and 19% below control) whereas food consumption was reduced exclusively in females (up to 14% below control). Mean body weight was decreased in males and females of the high-dose (up to 22 and 14% below control) and females of the mid-dose group (up to 7% below control). Body weight change was decreased in both sexes of F0 high and mid-dose animals. Body weight decreases were 11 and 6% below controls in males and females of cohort 1A, 12 and 6% in males and females of cohort 1B. In almost all cases, body weight change was correspondingly decreased. The reduction in food and water consumption of high-dose F0 and mid-dose F0 animals in combination with the body weight decreases summed up to a treatment-related and adverse assessment. However, corresponding decreased terminal body weights of males and females were confirmed by necropsy. Therefore, this combination was assessed as treatment-related adverse.

All other parameters were not altered. No treatment-related adverse findings were observed in the low-dose (1.5 mg/kg bw/d) F0 animals.

Regarding clinical pathology, in F0 rats of both sexes in test group 3 (15 mg/kg bw/d) stress was indicated by decreased absolute and relative eosinophil cells in males, but by increased absolute and relative monocyte counts in females. In the mentioned individuals no liver parameters were relevantly changed apart from higher triglyceride levels in F0 males, but aspartate aminotransferase (AST) activities and inorganic phosphate levels were increased. These altered parameters in combination with lower body weights of the rats were most probably due to a retarded growth affecting bones (inorganic phosphate, ALP) as well as skeletal muscle (AST).

Regarding pathology, terminal body weight was significantly decreased in males (-22%) and females (-12%) of test group 3. They were below the historical control values and were therefore considered treatment-related. Consequently, significant absolute weight decreases and/or significant relative weight increases were seen in several organs, that were considered secondary to the decreased terminal body weight.

Histopathology revealed treatment-related findings in following target organs of males and females: brain, esophagus, eyes with optic nerve, glandular stomach, heart, kidneys, liver, lungs, axillary and mesenteric lymph nodes, pancreas, pituitary gland, and skeletal muscle. Furthermore, the adrenal cortex, left epididymis and seminal vesicles were affected only in male animals.

The main finding in all these organs was a “microvesicular” type of cytoplasmic vacuolation, characterized by the presence of very few to multiple vacuoles, ranging from very fine to small vacuoles (no larger than the nuclei of the cell). Characteristically, if the cytoplasmic vacuolation was abundant, the cells were very clear or pale and increased in size. Vacuoles larger than the cell nuclei were referred here as “macrovesicular” type of vacuolation, which was observed in few organs (brain and seminal vesicles). In 13 out of 16 target organs the vacuolation occurred alone without additional signs of cytotoxicity. Only in 3 out of 16 target organs (kidneys, liver and skeletal muscle) the vacuolation was associated with signs of cytotoxicity (degeneration/regeneration, inflammation and apoptosis/single cell necrosis).

A summary of the histopathological treatment-related findings is given as follows:

In test group 3 (15 mg/kg bw/d), the adrenal cortex of males showed a microvesicular vacuolation of the zona fasciculata (minimal to moderate), giving the cytoplasm of the cells a foamy and pale aspect and often causing an increased size of the cells. This finding might have contributed to the significant relative weight increase of the adrenal glands (+41%). Females were not affected. In the left epididymis, the vacuolation was localized in the ducts of approximately 2/3 of the distal corpus and never included the cauda. The vacuolation (minimal to moderate) ranged from small microvesicular vacuoles to vacuoles as large as the nuclei. In the seminal vesicles, the vacuolation of the epithelial cells (minimal to moderate) was characterized by small vacuoles of very regular size localized at the basal part of the cells displacing the nuclei to the apical pole. Single vacuoles of macrovesicular type were also present in the most severe cases.

In following organs of test group 3, males and females were affected:

In the axillary and mesenteric lymph nodes, small vacuoles (minimal to slight) were seen in the cells of the high endothelial venules (HEV). In the brain, a microvesicular and macrovesicular vacuolation (minimal to moderate) was noted in the choroid plexus of the lateral and dorsal third ventricles. Males were more severely affected than females. In the esophagus, the vacuolation was seen in the skeletal muscle of the wall (minimal to slight), with similar characteristics as observed in the gastrocnemius muscle: very tiny, transparent to birefringent microvacuoles ranging approximately from 2 – 4μm in diameter within the fibers. A very fine vacuolation in the eyes (minimal to slight) was localized in the retinal pigment epithelium. The affected cells displayed a ground glass aspect and were minimally enlarged. In the glandular stomach, the vacuolation characterized by small vacuoles (minimal to slight) were observed at the base of the glands in the pyloric mucosa. The heart showed a microvesicular vacuolation affecting mainly the septum and left ventricular wall. This change was not associated with degeneration or necrosis but conferred the cells a damaged aspect. In the kidneys the vacuolation was observed in the medulla (tubules of the inner stripe of the outer medulla) in males and females, whereas only in males, degeneration/regeneration was noted in the cortex (proximal convoluted tubules) and was minimal to moderate. The vacuolation in the medulla was of a very fine microvesicular type (minimal to slight), giving the epithelial cells a “ground glass” pale and larger aspect. The more severe findings in the kidneys of males might have contributed to significantly increase the relative weight (+31%). The degeneration/regeneration was characterized by multifocal microvesicular vacuolation with loss of normal architecture due to nuclear disorganization and crowding, single pyknosis and general light basophilia. In the liver, the vacuolation in the hepatocytes was characterized by very small vacuoles of regular size (foamy aspect), which was more severe in females (minimal to moderate) than males (minimal to slight). An associated mixed-cell inflammation (minimal to moderate) and multinucleated hepatocytes were observed particularly in females and not in males. This type of inflammation was often associated with apoptosis/single cell necrosis (minimal to slight). Furthermore, the epithelium of the portal bile ducts of large caliber also showed a vacuolation with a ground-glass appearance in both sexes. In the lungs, the vacuolation (minimal to slight) was localized in the bronchial and bronchiolar epithelium and was characterized by a “foamy” aspect. In the bronchial associated lymphoid tissue (BALT), the high endothelial venules (HEV) showed the same type of vacuolation (minimal) as described in the lymph nodes. In the pancreas, a microvesicular vacuolation of the acinar epithelium was diffusely distributed along the organ (minimal to slight). Furthermore, a vacuolation was seen in the interlobular pancreatic ducts, presenting a ground glass appearance of the epithelial cells accompanied by increase size (minimal to slight). In the pituitary gland, the vacuolation was localized in the cells of the pars distalis. Within the cells the vacuolation was of a very fine microvesicular type, giving the cells a fine “granular” and pale aspect. It remains uncertain, if the vacuolation in the pituitary gland of females might have contributed to the significant relative weight increase of +41%. In the skeletal muscle (gastrocnemius muscle), as already described for the skeletal muscle of the esophageal wall, the vacuolation was characterized by very tiny microvacuoles with a birefringent aspect, ranging approximately from 2 – 4μm in diameter, with a multifocal distribution pattern within the muscle fiber. The vacuolation was minimal to slight in males and minimal in females. In addition, degeneration and /or regeneration of the myofibers was noted in males and females.

In test group 2 (5 mg/kg bw/d), the incidence and/or grading of the treatment-related vacuolation was generally lower and was not associated with additional signs of cytotoxicity. Organs showing vacuolation in males only were: axillary and mesenteric lymph nodes, heart, left epididymis, pituitary gland and seminal vesicles. Organs with treatment-related vacuolation affecting both males and females were: esophagus, glandular stomach, kidneys, lungs, pancreas and skeletal muscle.

In test group 1 (1.5 mg/kg bw/d), no treatment-related findings were noted.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered incidental or spontaneous in origin and without any relation to treatment.

The morphological pattern and abundant distribution of the vacuolation in 16 target organs suggests that this finding represents most likely a systemic phospholipidosis (Nonoyama and Fukada, 2008; Rudmann et al, 2004). In a previous study (supporting study below, BASF project No.: 85R0695/00R047) with the same test substance, special stains (PAS, Oil-Red-Oil and Sudan black) revealed that the vacuolation most likely had a phospholipidic nature. Since a definitive diagnosis of phospholipidosis should rely on transmission electron microscopy or chemical analysis, a conclusive diagnosis for the presence of phospholipidosis cannot be done in this study. However, based on the systemic distribution of the vacuolation affecting organs involved in multiple vital functions (brain, eyes, heart, etc.), the cytoplasmic vacuolation associated or not with cytotoxicity in in males and females of test groups 3 and 2 was assessed as treatment-related and adverse.

3. Supporting study (OECD TG 422):

In a study according OECD TG 422 the substance was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 1.5, 5.0 and 15 mg/kg bw/day (BASF SE, 2019). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (0.5% Sodium carboxymethyl cellulose [0.5% CMC] suspension in drinking water). The duration of treatment covered a 2-week pre-mating and a mating period, approximately 1 day post-mating in males, and the entire gestation period as well as approximately 22 days of the lactation period in females.

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females.

In this study signs of systemic toxicity at the highest dose of 15 mg/kg bw/d such as a reduction in food consumption and decrease in body weight (change), adverse changes in clinical pathology parameters and histopathology have been reported. Mid-dose males and females showed only minor, temporary and inconsistent changes in water consumption, food consumption and body weight during the whole study period which were assessed to be insufficient to proof adversity.

Regarding pathology, target organs were the liver, brain, axillary and mesenteric lymph nodes and the pyloric mucosa of the glandular stomach.

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.

No test-item related effects on oestrus cycle, sperm measures and reproductive performance were observed and therefore, the NOAEL for reproductive performance and fertility was set to 15 mg/kg bw/d for male and female Wistar rats. Furthermore, no treatment-related effects on the foetuses up to PND 13 were detected and the NOAEL for developmental toxicity was 15 mg/kg bw/d, the highest dose tested.

A vacuolization of the organs (liver, brain, stomach, lymph nodes) was also detected in that study and these changes are assumed to contribute to a treatment-related phospholipidosis. In females of test group 3 (15 mg/kg bw/d) in the OECD TG 422 study the relative liver weight was statistically significantly increased (3.018%). Although this weight increase was within the historical control range (2.173 – 3.312%) a treatment-related vacuolation of the hepatocytes might have contributed to the weight increase. The vacuolation was characterized by a hepatocellular microvesicular cytoplasmic pattern, that was centrilobular in males (minimal to slight) and diffuse in females (minimal to moderate) at 15 mg/kg bw/d. Special stains performed in the liver to better characterize the nature of this vacuolation, excluded the presence of glycoproteins (PAS negative) or a fatty change (Oil-Red-O stained negative for neutral fat) suggesting that the vacuolation might be of a phospholipidic nature (Sudan Black stain positive together with Oil- Red-O negative stain). The same microvesicular pattern of vacuolation was observed in epithelial cells of other organs of male and female animals of test group 3, such as choroid plexus in the brain, high endothelial venules (HEV) in the axillary and mesenteric lymph nodes, and epithelial cells at the base of the glands of the pyloric mucosa. These changes, which were mostly minimal or slight, might also contribute to assume a treatment-related phospholipidosis. However, the definitive diagnosis is based on either phospholipid analysis or transmission electron microscopy as golden standard. Therefore, a conclusive diagnosis for the presence of phospholipidosis cannot be done in this study. Although no inflammatory or degenerative changes accompanied the vacuolation in examined organs, the presence of a fine vacuolation in the mentioned organs of males and females of test group 3 (15 mg/kg bw/d) was assessed as adverse.

Final discussion:

Repeated oral exposure to DMDC leads to systemic toxicity, basically reflected in reduced water and food consumption, mean body weight decrease, lower body weight change, adverse changes in clinical pathology parameters and histopathology. The latter is mainly characterised by an cytoplasmaic vacuolation in combination with the presence of very few to multiple vacuoles, ranging from very fine to small vacuoles. The vacuolization in various organs (liver, kidney, heart) observed in the OECD TG 408 study in the high dose animals (60 mg/kg bw/day) can be interpreted as treatment-related adverse effect. In liver and heart of the treated high dose animals, there was a degeneration observed, which can be considered a cytotoxic effect. However, neither inflammation nor necrosis has been detected which could be correlated with the vacuolization. The content of the vacuoles was free of neutral fat and glycogen. No electron microscopic imaging or lipid analysis was performed to characterize the vacuoles in more detail. Therefore, a conclusive diagnosis of the nature of this effect cannot be done in this study.

In the high dose group of the OECD TG 443 there were histopathological revealed treatment-related findings (vacuolation) in following target organs of males and females: brain, esophagus, eyes with optic nerve, glandular stomach, heart, kidneys, liver, lungs, axillary and mesenteric lymph nodes, pancreas, pituitary gland, and skeletal muscle. Furthermore, the adrenal cortex, left epididymis and seminal vesicles were affected only in male animals. In 13 out of 16 target organs the vacuolation occurred also alone without additional signs of cytotoxicity. However, in 3 out of 16 target organs (kidneys, liver and skeletal muscle) the vacuolation was associated with signs of cytotoxicity (degeneration/regeneration, inflammation and apoptosis/single cell necrosis).

In the mid-dose groups of both studies (OECD TG 408: 12 mg/kg bw; OECD TG 443: 5 mg/kg bw) vacuolation was also observed, but not in the presence of cell damage. The lowest dosages were free of adverse effects (OECD TG 408: 2.5 mg/kg bw; OECD TG 443: 1.5 mg/kg bw).

According to the CLP, specific target organ toxicity (STOT RE) is defined as a specific, target organ toxicity arising from a repeated exposure to a substance or mixture. All significant health effects that can impair function, both reversible and irreversible, immediate and/or delayed are included. These adverse health effects include consistent and identifiable toxic effects in humans, or, in experimental animals, toxicologically significant changes which have affected the function or morphology of a tissue/organ. Although the NOAELs of all discussed studies are in the low dose groups, only the high dose groups show adverse effects in combination with functional changes of a tissue such as degeneration/regeneration, inflammation and apoptosis/single cell necrosis. The high dose groups of both WoE studies are beyond 10 mg/kg bw, which is the limit between STOT cat. 2 and cat. 1. Based on the findings in all studies, DMDC is therefore classified as STOT RE cat. 2 (target organs: kidney, liver, skeletal muscle, heart). Nevertheless, the NOEALs used for DNEL derivation were those of the low dose groups. As a worst case, as the starting point for DNEL derivation the lowest NOAEL was used: 1.5 mg/kg bw (OECD TG 443).

Inhalation

In a subchronic inhalation study (OECD TG 413 and GLP), rats were exposed to aerosol concentrations of 0, 2, 12 and 48 mg/m³ for 3 months (6 hours/day and 5 days/week) (BASF AG, 1992). No mortalities occurred. In the high exposure group local irritative effects, typical for alkaline compounds such as amines were observed for the skin (slight hyperkeratosis in 7/10 animals) and upper airways (nasal mucosa, slight vacuolization of olfactory epithelium in 2/10 high dose males, and in 1/10 high dose females). A clear and statistically significant depression of body weight development was noted in animals of both sexes. Compared to control animals terminal body weight was significantly reduced by 14 % in males (p < 0.01) and 8 % in females (p < 0.05). Systemic toxicity was mild. Relative organ weight of liver, lung, and kidney was significantly increased in high dose male and female animals on the 1 % or 5 % level of significance. Relative weight of adrenals (p < 0.05) and testes (p < 0.01) and absolute lung weight (1.41 g vs. 1.18 g in controls, p < 0.05) were significantly increased only in high dose male rats. The relative organ weight changes were largely influenced by reduced body weights and were judged to be of minor relevance. Pathological correlates were not found for any of these organs, and histological alterations in the testes were not seen.

The liver was also a target organ in high dose male rats, but not in high dose females, as substantiated by significant increases of serum transaminases GOT and GPT (glutamate oxalo-acetate transaminase and glutamate pyruvate transaminase, both on the p < 0.01 level). Activity of GPT in serum was 1.081 μkat/L in high dose male rats compared to 0.845 μkat in control animals. However, no histopathological correlate was seen. Red blood cells were affected in high dose male rats as substantiated by significant reductions (p < 0.05) of hemoglobin, hemoglobin per erythrocyte, mean corpuscular hemoglobin concentration, and polychromatosis. In spleen hemosiderin was noted in all high dose animals and extramedulary haematopoesis (9/10 high dose females) was indicative of a mild anemic effect. A test substance related effect on kidneys was of borderline significance (slight tubular nephrosis in 6/10 high dose males vs. 1/10 male controls; in females 7/10 mid dose and 9/10 high dose rats vs. 7/10 control animals) with increased relative kidney weights (p < 0.01) and increased urea concentration in females (p < 0.01; unchanged in males). In the mid dose animals only a marginal yet significant increase of GPT and alkaline phosphatase levels (both at p < 0.05) in the male rats were seen. Alkaline phosphatase (AP) was not significantly increased in animals at the higher dose level. Therefore no dose-relation was given for AP, and this finding was not regarded as a treatment-related effect. The increase of GPT in mid dose males was marginal (1.043 μkat/L vs. 0.845 μkat/L in controls). GOT (glutamateoxaloacetate transaminase) was not affected in this animal group. No substance-related effect was noted in the low dose groups.

The marginal increase in GPT level at 12 mg/m3 was not considered toxicologically relevant in the absence of an increase in GOT levels and any other effects in the liver. Therefore, the NOAEC was set at 12 mg/m3.

In conclusion, repeated inhalation of the test item showed systemic toxicity in the target organs liver and spleen in the form of hepatotoxicity and a disturbed hemoglobin metabolism. The amine-specific local toxicity was manifest in the form of changes in the target organs skin and nasal mucosa and led to hyperkeratosis or to a degeneration of the olfactory epithelium. A scleroderma described in the literature after intraperitoneal administration or dermal application was not detected under the test conditions chosen. Only a local effect on the skin or mucosa was detected as the result of an impaction of the aerosol or of a sorption of the vapors.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. The substance is considered to be classified for repeated dose toxicity STOT RE cat.2 (H373, target organs: liver, kidney, skeletal muscle, heart) according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.