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EC number: 239-763-1 | CAS number: 15680-42-9
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Toxicity to reproduction
Administrative data
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2014-2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Cross-reference
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2014 - 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to other study
- Remarks:
- Dose-Range-Finder
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 11-12 weeks
- Weight at study initiation: 220 g (females) and 310g (males)
- Fasting period before study: no
- Housing: individually, following exceptions: During overnight matings, male and female mating partners were housed together. Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7d
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: October 27th 2014 To:December 22nd 2014 - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Remarks:
- suspension
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: weekly
- applied as a suspension - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The stability of the test substance in corn oil was demonstrated over a period of 7 days at room temperature.
- Duration of treatment / exposure:
- The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males,
and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females. - Frequency of treatment:
- daily
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Range finder test with 300 and 1000 mg/kg bw (project no. 1OC0416/13C103), no effects up to 1000 mg/kg bw other than dose-dependent increase in copper content in livers. Adverse findings on liver histopathology in one of 8 animals at 1000 mg/kg bw could not be clearly attributed to treatment and were not reason enough to lower the highest dose for the main study. Parameters investigated were body weight, food consumption, clinical chemistry parameters, haematology and liver histopathology. The study has been entered in the section on toxicokinetics (IUCLID 7.1.1).
- Rationale for animal assignment (if not random): Randomization - Positive control:
- no
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
FOOD CONSUMPTION
- weekly
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 4
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the course of FOB
- Dose groups that were examined: all
HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of administration period
- Anaesthetic used for blood collection: Yes, anaesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany)
- Animals fasted: Yes
- How many animals: 5/sex/dose
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of administration period
- Animals fasted: Yes
- How many animals: 5/sex/dose
URINALYSIS: Yes
- Time schedule for collection of urine: males: after mating, females: 1 day before end of administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: male: postnatal day 0, female: 10d after gestation
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
1. Adrenal glands
2. All gross lesions
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal gland
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Target organs
43. Testes (modified Davidson’s solution)
44. Thymus
45. Thyroid glands
46. Trachea
47. Urinary bladder
48. Uterus
49. Vagina
HISTOPATHOLOGY: Yes, control and high dose group, gross lesions in all animals
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Liver
16. Lung
17. Lymph nodes (mesenteric and axillary lymph nodes)
18. Ovaries
19. Oviducts
20. Peyer’s patches
21. Prostate
22. Rectum
23. Sciatic nerve
24. Seminal vesicles
25. Spinal cord (cervical, thoracic and lumbar cords)
26. Spleen
27. Stomach (forestomach and glandular stomach)
28. Testes
29. Thymus
30. Thyroid glands
31. Trachea
32. Urinary bladder
33. Uterus
34. Vagina - Other examinations:
- Staining for copper of liver histopathology samples.
- Statistics:
- Blood parameters:
For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose
group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians
For parameters with unidirectional changes:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
Urinalysis parameters: WILCOXON-test (one-sided)
Food consumption: DUNNETT-test (twosided)
fertility indices: FISHER'S EXACT test
Proportions of affected pups per litter with necropsy observations: WILCOXON-test
Weight parameters: KRUSKAL-WALLIS test - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- salivation post dosing
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- salivation post dosing
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- no effects observed
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- In males of test group 3 (1000 mg/kg bw/d) total white blood cell (WBC) counts and absolute lymphocyte counts were increased.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Males: ALT,total bile acids, cholesterol increased
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- The content of the digestive tract showed a green discoloration. This finding was regarded to be caused by the test substance, a greenishyellow pigment.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- liver, see figure
- Histopathological findings: neoplastic:
- no effects observed
- Details on results:
- The concentrations in corn oil were found to be in the range of 97 to 107% of the nominal concentration. Analyses demonstrated a recovery of 106% in the low dose (100 mg/kg bw/d), 105% in the mid dose (300 mg/kg bw/d) and 98% in the high dose (1000 mg/kg bw/d). Homogeneity of the test substance preparations was also demonstrated.
No parental animal died or was sacrificed moribund during the study period.
Green discolored feces were seen in all animals of the test groups 2 and 3 (300 and 1000 mg/kg bw/d). The sign came up first on study day 1 in test group 3 (1000 mg/kg bw/d) and study day 8 in test group 2 (300 mg/kg bw/d).
This green discoloration was regarded to be caused by the test substance, a greenishyellow pigment. It was regarded to be test substance related but not adverse. Furthermore, slight to severe salivation was observed during pre-mating in 8 males and 6 females in test group 3 (1000 mg/kg bw/d). Likewise during mating in 5 males in test group 2 (300 mg/kg bw/d), 9 males and 8 females in test group 3 (1000 mg/kg bw/d).
During post-mating slight to severe salivation was observed in 6 males in test group 2 (300 mg/kg bw/d) and 9 males in test group 3 (1000 mg/kg bw/d). The findings were considered to be related to treatment, most likely caused by bad taste of the test compound, and therefore assessed as being not adverse.
No adverse effects were noted in the functional observation battery.
For males of test group 3 (1000 mg/kg bw/d) total white blood cell (WBC) counts and absolute lymphocyte counts were increased.
At the end of the administration period in males of test groups 2 and 3 (300 and 1000 mg/kg bw/d) alanine aminotransferase (ALT) activities, total bile acid (TBA) and cholesterol levels were increased (ALT in test group 3 not statistically significantly increased). Cholesterol levels were already higher in males of test group 1 (100 mg/kg bw/d) although not statistically significantly. However, in this test group this was the only alteration and therefore it was regarded as treatment-related but not adverse (ECETOC Technical Report No 85, 2002). Additionally, in males of test group 3 (1000 mg/kg bw/d) total bilirubin levels were increased.
In the livers of test group 3 males and females (1000 mg/kg bw/day) increased numbers of single cell necrosis (normal or enlarged hepatocellular size, light eosinophilic, granular cytoplasm, often no nucleus detectable) or apoptosis was observed. This was also observed in one male animal of test group 2 (300 mg/kg bw/day). Furthermore, there was an increase in Kupffer cells, which occasionally formed aggregates. The Kupffer cells often
were of larger size compared to control animals. These findings were mainly observed in the left lateral lobe.
With the rubeanic acid stain greenish to black particles, most probably copper, located within the cytoplasm of hepatocytes of the treated animals but not the control animals could be detected. - Dose descriptor:
- NOEL
- Effect level:
- 100 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- System:
- hepatobiliary
- Organ:
- liver
- Treatment related:
- yes
- Dose response relationship:
- yes
- Conclusions:
- The substance shows adverse effects on rat liver at high doses. These effects appear to be related to accumulation of copper.
Table 1: Total white and differential blood cell count in males at the end of the in-life phase
0 mg/kg bw/d | 100 mg/kg bw/d | 300 mg/kg bw/d | 1000 mg/kg bw/d | ||
WBC | Mean | 4.50 v | 4.76 | 4.61 | 6.44 * |
[giga/L] | S.d. | 0.77 | 0.56 | 0.81 | 1.36 |
day 42 | N | 5 | 5 | 5 | 5 |
Median | 4.55 | 4.54 | 4.52 | 5.74 | |
NEUTA | Mean | 0.73 k | 1.10 | 1.07 | 1.16 |
[giga/L] | S.d. | 0.22 | 0.53 | 0.24 | 0.22 |
day 42 | N | 5 | 5 | 5 | 5 |
Median | 0.63 | 1.00 | 0.98 | 1.15 | |
LYMPHA | Mean | 3.58 v | 3.45 | 3.33 | 5.02 * |
[giga/L] | S.d. | 0.58 | 0.33 | 0.64 | 1.27 |
day 42 | N | 5 | 5 | 5 | 5 |
Median | 3.51 | 3.41 | 3.52 | 4.50 | |
MONOA | Mean | 0.08 k | 0.10 | 0.11 | 0.12 |
[giga/L] | S.d. | 0.04 | 0.02 | 0.05 | 0.02 |
day 42 | N | 5 | 5 | 5 | 5 |
Median | 0.09 | 0.11 | 0.10 | 0.12 | |
EOSA | Mean | 0.08 k | 0.08 | 0.08 | 0.10 |
[giga/L] | S.d. | 0.02 | 0.03 | 0.03 | 0.03 |
day 42 | N | 5 | 5 | 5 | 5 |
Median | 0.08 | 0.08 | 0.07 | 0.11 | |
BASOA | Mean | 0.01 k | 0.01 | 0.01 | 0.01 |
[giga/L] | S.d. | 0.00 | 0.01 | 0.00 | 0.01 |
day 42 | N | 5 | 5 | 5 | 5 |
Median | 0.01 | 0.01 | 0.01 | 0.01 | |
LUCA | Mean | 0.02 v | 0.02 | 0.01 | 0.03 |
[giga/L] | S.d. | 0.01 | 0.01 | 0.01 | 0.01 |
day 42 | N | 5 | 5 | 5 | 5 |
Median | 0.02 | 0.02 | 0.01 | 0.03 | |
NEUT | Mean | 16.1 k | 22.5 | 23.4 | 18.4 |
[%] | S.d. | 3.2 | 8.4 | 4.3 | 4.2 |
day 42 | N | 5 | 5 | 5 | 5 |
Median | 15.4 | 23.2 | 23.2 | 17.4 | |
LYMPH | Mean | 79.8 k | 73.2 | 72.1 | 77.5 |
[%] | S.d. | 4.1 | 8.3 | 4.1 | 4.2 |
day 42 | N | 5 | 5 | 5 | 5 |
Median | 79.9 | 72.5 | 72.7 | 78.4 | |
MONO | Mean | 1.8 k | 2.1 | 2.2 | 1.9 |
[%] | S.d. | 0.8 | 0.5 | 0.7 | 0.4 |
day 42 | N | 5 | 5 | 5 | 5 |
Median | 1.7 | 2.1 | 2.1 | 1.8 | |
EOS | Mean | 1.7 k | 1.7 | 1.8 | 1.6 |
[%] | S.d. | 0.4 | 0.4 | 0.5 | 0.5 |
day 42 | N | 5 | 5 | 5 | 5 |
Median | 1.6 | 1.8 | 1.9 | 1.5 | |
BASO | Mean | 0.2 k | 0.1 | 0.2 | 0.2 |
[%] | S.d. | 0.1 | 0.1 | 0.0 | 0.0 |
day 42 | N | 5 | 5 | 5 | 5 |
Median | 0.2 | 0.1 | 0.2 | 0.2 | |
LUC | Mean | 0.4 k | 0.4 | 0.2 | 0.4 |
[%] | S.d. | 0.2 | 0.1 | 0.1 | 0.1 |
day 42 | N | 5 | 5 | 5 | 5 |
Median | 0.3 | 0.4 | 0.2 | 0.3 |
Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics
v=KRUSKAL-WALLIS-WILCOX; k=KRUSKAL-WALLIS
Table 2: Enzymes in males at the end of the in-life phase
0 mg/kg bw/d | 100 mg/kg bw/d | 300 mg/kg bw/d | 1000 mg/kg bw/d | ||
ALT | Mean | 0.64 v | 0.61 | 1.13 ** | 1.47 |
[μkat/L] | S.d. | 0.09 | 0.06 | 0.37 | 0.91 |
day 42 | N | 5 | 5 | 5 | 5 |
Median | 0.61 | 0.58 | 1.02 | 1.08 | |
AST | Mean | 1.50 k | 1.48 | 2.03 | 2.67 |
[μkat/L] | S.d. | 0.21 | 0.31 | 0.61 | 1.38 |
day 42 | N | 5 | 5 | 5 | 5 |
Median | 1.48 | 1.40 | 1.90 | 1.87 | |
ALP | Mean | 1.40 k | 1.38 | 1.78 | 1.44 |
[μkat/L] | S.d. | 0.30 | 0.27 | 0.53 | 0.48 |
day 42 | N | 5 | 5 | 5 | 5 |
Median | 1.27 | 1.41 | 1.89 | 1.62 | |
GGT_C | Mean | 7 k | 7 | 8 | 8 |
[nkat/L] | S.d. | 3 | 3 | 4 | 5 |
day 42 | N | 5 | 5 | 5 | 5 |
Median | 8 | 9 | 9 | 7 |
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 015
- Report date:
- 2015
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
Test material
- Reference substance name:
- [1-[[(2-hydroxyphenyl)imino]methyl]-2-naphtholato(2-)-N,O,O']copper
- EC Number:
- 239-763-1
- EC Name:
- [1-[[(2-hydroxyphenyl)imino]methyl]-2-naphtholato(2-)-N,O,O']copper
- Cas Number:
- 15680-42-9
- Molecular formula:
- C17H11CuNO2
- IUPAC Name:
- [1-[[(2-hydroxyphenyl)imino]methyl]-2-naphtholato(2-)-N,O,O']copper
- Test material form:
- solid: nanoform
- Details on test material:
- - Substance type: pigment
- Physical state: Solid / greenish yellow
- Name of test material (as cited in study report): Irgazin Yellow L 0800
- Lot/batch No.: 0005629903
- Analytical purity: 99.0 %
- Purity test date: 2013
- Expiration date of the lot/batch: unlimited stability under storage conditions
- Stability under test conditions: stable
- Storage condition of test material: room temperature
- Date of manufacture: 01 Dec 2011
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 11-12 weeks
- Weight at study initiation: 220 g (females) and 310g (males)
- Fasting period before study: no
- Housing: individually, following exceptions: During overnight matings, male and female mating partners were housed together. Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7d
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: October 27th 2014 To:December 22nd 2014
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: weekly
- applied as a suspension (4mL/kg bw - Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: maximum 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): pregnant animals and litter together - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The stability of the test substance in corn oil was demonstrated over a period of 7 days at room temperature.
- Duration of treatment / exposure:
- The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males,
and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females. - Frequency of treatment:
- daily
- Details on study schedule:
- - Age at mating of the mated animals in the study: 14-15 weeks
Doses / concentrationsopen allclose all
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: range finder test 300 and 1000 mg/kg bw (project no. 1OC0416/13C103), no effects up to 1000 mg/kg bw
- Rationale for animal assignment (if not random): Randomization - Positive control:
- no
Examinations
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
FOOD CONSUMPTION
- weekly
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 4
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the course of FOB
- Dose groups that were examined: all
HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of administration period
- Anaesthetic used for blood collection: Yes, anaesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany)
- Animals fasted: Yes
- How many animals: 5/sex/dose
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of administration period
- Animals fasted: Yes
- How many animals: 5/sex/dose
URINALYSIS: Yes
- Time schedule for collection of urine: males: after mating, females: 1 day before end of administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: male: postnatal day 0, female: 10d after gestation
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity - Sperm parameters (parental animals):
- stages of spermatogenesis were examined in histopathology
- Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality up to day 4, presence of gross anomalies
GROSS EXAMINATION OF DEAD PUPS:
yes, all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically - Postmortem examinations (parental animals):
- GROSS PATHOLOGY: Yes
1. Adrenal glands
2. All gross lesions
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal gland
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Target organs
43. Testes (modified Davidson’s solution)
44. Thymus
45. Thyroid glands
46. Trachea
47. Urinary bladder
48. Uterus
49. Vagina
HISTOPATHOLOGY
ORGAN WEIGHTS: Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus
HISTOPATHOLOGY: Yes, control and high dose group, gross lesions in all animals
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Liver
16. Lung
17. Lymph nodes (mesenteric and axillary lymph nodes)
18. Ovaries
19. Oviducts
20. Peyer’s patches
21. Prostate
22. Rectum
23. Sciatic nerve
24. Seminal vesicles
25. Spinal cord (cervical, thoracic and lumbar cords)
26. Spleen
27. Stomach (forestomach and glandular stomach)
28. Testes
29. Thymus
30. Thyroid glands
31. Trachea
32. Urinary bladder
33. Uterus
34. Vagina - Postmortem examinations (offspring):
- SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: All pups delivered from the F0 parents were examined as soon as possible on the day of birth
GROSS NECROPSY
All surviving pups (sacrificed on PND 4 under isoflurane anesthesia with CO2), all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically. - Statistics:
- Blood parameters:
For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose
group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians
For parameters with unidirectional changes:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
Urinalysis parameters: WILCOXON-test (one-sided)
Food consumption: DUNNETT-test (twosided)
fertility indices: FISHER'S EXACT test
Proportions of affected pups per litter with necropsy observations: WILCOXON-test
Weight parameters: KRUSKAL-WALLIS test - Reproductive indices:
- Male reproduction data:
- Male mating index
- Male fertility index
Female reproduction and delivery data:
- Female mating index
- Female fertility index
- Gestation index
- Live birth index
- Post implantation loss - Offspring viability indices:
- Pup number and status at delivery
Pup viability/mortality
Sex ratio
Pup body weight data
Results and discussion
Results: P0 (first parental generation)
General toxicity (P0)
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- salivation post dosing
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- liver
Reproductive function / performance (P0)
- Reproductive function: oestrous cycle:
- not examined
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
Details on results (P0)
produce F1 litter. One male of test group 1 (No. 16 mated with female No. 116) and one male in test group 2 (No. 23 mated with female No. 123) did not generate implants in utero. The male fertility index was 100% in test groups 0 and 3 and 90% in test groups 1 and 2.
The female mating index calculated after the mating period for F1 litter was 100% in all
test groups.
The mean duration until sperm was detected (GD 0) was 1.7, 2.7, 2.5, 2.1 days in test groups 0 - 3. This finding reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
All sperm positive rats got pregnant with one exception in test group 1 and one exception in test group 2. Female animal No. 116 (test group 1) which was mated with male No. 16 and female No. 123 which was mated with male No. 23 did get sperm in vaginal smear but showed no implants. The female fertility index was 100% in test groups 0 and 3 and 90% in test groups 1 and 2.
The mean duration of gestation was similar in all test groups (22.2 days in test group 0; 22.1 days in test groups 1 and 2, 22.3 days in test group 3). These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
The gestation index was 100% in test group 0, 1 and 3 and 89% in test group 2 (300 mg/kg bw/d). The latter represents 8 females with live pups on the day of birth out of 9 females pregnant. Also in the historical control data not always 100% of the pregnant females had live pups: the historical control data ranges from 89% to 100%..
Effect levels (P0)
open allclose all
- Dose descriptor:
- NOEL
- Remarks:
- fertility
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No adverse effects on fertility at the limit dose
- Dose descriptor:
- NOEL
- Remarks:
- general toxicity
- Effect level:
- 100 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
Target system / organ toxicity (P0)
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- System:
- hepatobiliary
- Organ:
- liver
- Treatment related:
- yes
- Dose response relationship:
- yes
Results: F1 generation
General toxicity (F1)
- Clinical signs:
- no effects observed
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- not examined
Details on results (F1)
The postimplantation loss was 16.5% in test group 0, 4.8% in test group 1 (100 mg/kg bw/d), 12.7% in test group 2 (300 mg/kg bw/d) and 7.3% in test group 3 (1000 mg/kg bw/d).
These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range (0.7% - 18.3%) of the historical control data.
The viability index indicating pup mortality during lactation (PND 0 - 4) was 96.7% (test group 0), 100% (test group 1, 100 mg/kg bw/d), 98.4% (test group 2) and 95.6% (test group 3). The indices were based on two pups of test group 3 (1000 mg/kg bw/d) and one pup in test group 1 (100 mg/kg bw/d) cannibalized and two pups (test group 2) and three pups (test group 3) found dead. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show significant differences between the control and the test substance-treated groups.
In test group 1 (100 mg/kg bw/d) one and test group 3 (1000 mg/kg bw/d) five female pups of one dam (animal no. 131) showed a discolored thymus. The color was described as white instead of gray. In test group 2 (300 mg/kg bw/d) one female pup showed a discolored liver (pale) and its thorax was fluid filled (blood coagulum). These findings were assessed as being signs occurred post mortem or spontaneous in nature and thereby not related to treatment.
Effect levels (F1)
- Dose descriptor:
- NOEL
- Remarks:
- developmental toxicity
- Generation:
- F1
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Parameters up to postnatal day 4 (screening study)
Target system / organ toxicity (F1)
- Critical effects observed:
- no
Overall reproductive toxicity
- Reproductive effects observed:
- no
Applicant's summary and conclusion
- Conclusions:
- The substance caused no adverse effects on fertility and offspring development in a screening study (OECD 422) in rats with gavage application of 100, 300 and 1000 mg/kg bw.
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