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EC number: 269-284-3 | CAS number: 68214-04-0
- 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
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
In a test according to OECD 422 the substance was administered daily by oral gavage to 12 male and female adult rats in three groups at dosages of 500 mg (1000 mg during study days 1-28), 250 mg and 62,5 mg test item per kg body weight over a period of at least 28 days (males) and at least 49 days (females). Male and female animals were mated within their treatment group during the treatment period. Litters were used for evaluation of developmental and reproduction related parameters but did not receive test item treatment. Further 24 animals (12 males and 12 females) received the vehicle solution as control; all other parameters were identical.
Clinical signs and viability were monitored daily during the in-life phase. Individual body weight was recorded once weekly. Individual food / water consumption of female animals and group food / water consumption of male animals were recorded once weekly. Detailed clinical signs were monitored weekly from all adult animals. Grip strength and reactivity to stimuli (beam-walking test) was assessed from 5 randomly selected adult animals per sex and group during the last exposure week.
At the end of the treatment period, blood samples were taken from all adult animals to provide T4-hormone data. For assessment of additional toxicological parameters, 5 adult animals per sex and group were randomly selected for additional blood sampling (females during end of pre-mating, males at day of necropsy) to provide data on hematology and clinical biochemistry.
All adult and offspring animals were sacrificed after bleeding and examined by gross necropsy. Weights of selected organs were recorded and selected tissues and organs were preserved with special emphasis on organs of the reproductive system. From 5 randomly selected adult animals per sex and group, additional organs were preserved and examined microscopically.
Offspring parameters were assessed from each litter on day 0 post partum (pp), day 4pp, day 13pp and day of necropsy as specified by the study plan. These included numbers, stillbirth, live birth and runts, mortality, sex and presence of gross abnormalities. Blood samples from selected offspring per litter were taken on day 4pp to provide T4-hormone data.
The results of the repeated dose part can be found under repeated dose toxicity.
Regarding reproduction and development (achievement and duration of pregnancy, offspring losses, survival rate of pups and development of pups) no difference was observed for all test item treated-animals, when compared to the vehicle control animals. There was no evidence for any test item-related effect.
Conclusion:
A daily oral administration of the substance to male and female Wistar rats at dose levels of 500 mg (1000 mg during study days 1-28), 250 mg and 62,5 mg/kg body weight over a time period of 47-72 days for males and 51-91 days for females resulted in some minor animal behavioral changes observed in male and female animals predominately of the high dose groups, alterations in water consumption in male dose groups, changes in clinical biochemistry (AP lower and K higher) in the female high dose group and microscopic changes in the kidneys of male and female high dose animals. The findings were not considered adverse. As the findings could not be associated with a substantial impact on the animal’s health, the NOAEL regarding the repeated dose toxicity was set to 250 mg/kg body weight.
Furthermore, no pathological evidence for toxic effects on the reproduction performance of female and male rats was found. Regarding the overall time period of gestation/lactation and after birth until end of in-life phase no evidence for a toxic effect on the pup development could be detected. The NOAEL regarding reproduction and development of the substance under these study conditions was set to be 250 mg/kg body weight for the female and male animals.
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Remarks:
- according to OECD 422
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 29 August 2017 to 20 April 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- Wistar rats are commonly used and recommended to assess toxicity. A large number of publications on this subject are available as well as historic data and first-hand experience at the test facility.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- EST ANIMALS ,
- Source: Charles River, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Strain: Wistar Han (IGS) (Crl:WI(Han))
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 12-13 weeks
- Weight at study initiation: Males: 384-452 g; Females: 220-262 g
- Fasting period before study: none
- Housing: Males up to 3 in open macrolon cages type 2000P; Females (individually), paired animals
and single dams with their litters in open macrolon cages type III.
- Diet: Maintenance diet for rats and mice, No. 1324 TPF, ad libitum; Dams: Breeding diet for rats and mice No. 1314 TPF (Altromin Spezialfutter GmbH & Co. KG, 32791 Lage) ad libitum
- Water: sterilized tap water ad libitum)
- Acclimation period: 6-9 days (pre-treatment period for females 14 days)
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): ca 15/hour
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- autoclaved community tap water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: The substance was dissolved in water at concentrations of 256,8 (high dose until study day 28), 128,4 (high dose from study day 29), 64,2 and 16,1 mg/mL, corresponding to 200, 100, 50 and 12,5 mg/mL of Cmain. Since the test item is stable for 14 days at 5 ± 3°C , the test item solutions were prepared for up to 14 days in advance and were stored at 5 ± 3°C until day of application.
- VEHICLE : autoclaved community tap water
- dosing volume: 5 mL/kg bw - Details on mating procedure:
- - M/F ratio per cage: 1/1
- Length of cohabitation: minimum 7 days (based on one animal conceiving on day 7)
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings: 1 additional mating period for 4 females in the high dose group
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol:
The reproductive-success of the high dose group was below the minimal acceptable number of 8 pregnant females per group according to the guideline OECD422. Thus, 4 animals of the high dose group were included into a second mating phase with proven sires from the first mating period. For three animals a sperm plug was detected, all four animals achieved pregnancy. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The concentration of the substance in the respective solutions was determined once within pre-mating phase (02.10.2017), at the beginning of the gestation phase (16.10.2017) and at the end of gestation/ beginning of lactation phase (06.11.2017). Samples were analysed by HPLC/UV after storange and shipment at 5 ± 3°C.
Identification: HPLC_3
Components: Degasser G1379A; Quaternary pump G1311A; Autosampler G1313A; Column compartment G1316A; UV/VIS-Detector MWD G1365B
Manufacturer: Agilent Technologies
Software: CHROMELEON 6.80 SR11d, Build 3302
Solvent for stock solution: Demineralised water
Solvent for calibration: Demineralised water
Column: Dr. Maisch Reprosil Pur 120 C18-AQ, 5 µm, 150 x 4.6 mm
with precolumn Phenomenex SecurityGuard C18, 4 x 3 mm
Eluents: A: 50 mM ammonium acetate; B: Acetonitrile
Gradient: Time (min) % A % B
0.0 85 1 5
3.5 30 70
5 30 70
5.5 85 15
7.5 85 15
Flow rate: 1.5 mL/min
Column temperature: 40 °C
Detection: 512 nm
Injection volume: 10 µL
Retention time: 2.3 – 2.6 min
Calibration: 5 – 100 mg/L: y=0.17487x+0.00069 (R2=0.99998)
Recovery QC samples: 60 mg/L 99.8-100.4%; 50 mg/L 97.2-97.5%; 50 mg/L: 98.5-98.7% of nominal
LOQ 5 mg/L - Duration of treatment / exposure:
- Males:14 days prior to mating; during mating and 3-4 weeks post mating (total at least 47 days)
Females: 14 days prior to mating; during mating, during gestation and until day 13 of lactation (total at least 51 days)
For 4 pairs of the 1000/500 mg/kg bw group a second mating period was included. - Frequency of treatment:
- daily
- Dose / conc.:
- 500 mg/kg bw/day
- Remarks:
- Based on content of the chromophore (Cmain)
During day 1-28 1000 mg/kg bw was administered. This dose was lowered due to mortality of two animals in this group and one animal in the 250 mg/kg bw group showing signs of aspiration - Dose / conc.:
- 250 mg/kg bw/day
- Remarks:
- Based on content of the chromophore (Cmain)
- Dose / conc.:
- 62.5 mg/kg bw/day
- Remarks:
- Based on content of the chromophore (Cmain)
- No. of animals per sex per dose:
- 12 males + 12 females
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- Dose selection rationale: based on a dose range finding study
In a previously performed dose range finding study (DRF), a slightly different batch of the substance was administered in escalating doses up to 1055 mg/kg body weight over a time period of 21 days. The DRF showed mild adverse test item-related effect in terms of decrease of body weight in the female test animals at dose levels of 703 and 1055 mg/kg and signs of behavioral discomfort in both, male and female animals starting from 313 mg/kg. The dose levels referred to in the DRF are based on the main constituent (Cmain) of the test item. A different (slightly purer) batch of the substance was used for the main study. Since the OECD guideline 422 states, that the highest dose level should be chosen with the aim of inducing toxic effects but not death nor obvious suffering, the maximum dose for this study was based on the DRF data described above, showing no mortality or obvious suffering at the highest dose level. In this respect, the maximum dose of 1000 mg/kg was considered an acceptable high dose for this study. Two graduate (1 in 4) serial dilutions thereof (250 mg/kg, 62,5 mg/kg) were assigned to be medium and low dose
levels, respectively. - Positive control:
- NA
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for moratility, daily for clinical signs
DETAILED CLINICAL OBSERVATIONS: Yes in a standard arena
- Time schedule: weekly
Appearance (general status, physiology, autonomic functions, neurology, tonus); Motoric / exploration behavior; Excitation and Abnormal behavior
BODY WEIGHT: Yes
- Time schedule for examinations: At least once weekly (including once before beginning of application); during pregnancy, females were weighed individually on days 0, 7, 14 and 20 and within 24 hours of parturition (at birth: day 0 or 1 post partum) and on day 4, 9, 13 post-partum and on day of termination
FOOD CONSUMPTION: Yes
- Time schedule for examinations:
At least once weekly (including once before beginning of application); during pregnancy, females were weighed individually on days 0, 7, 14 and 20 and within 24 hours of parturition (at birth: day 0 or 1 post partum) and on day 4, 9, 13 post-partum and on day of termination
FOOD EFFICIENCY: not measured
WATER CONSUMPTION Yes
- Time schedule for examinations:
At least once weekly (including once before beginning of application); during pregnancy, females were weighed individually on days 0, 7, 14 and 20 and within 24 hours of parturition (at birth: day 0 or 1 post partum) and on day 4, 9, 13 post-partum and on day of termination
OPHTHALMOSCOPIC EXAMINATION:No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: males at termination; females after pre-mating
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: 5 males + 5 females
- Parameters checked: Leukocytes, Erythrocytes,Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin conc. (MCHC), Thrombocytes, Reticulocytes, Neutrophil granulocytes, Lymphocytes, Monocytes, Eosinophils, Basophils, blood clotting time
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: males at termination; females after pre-mating
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: 5 males + 5 females- Parameters checked: Alkaline phosphatase (AP), Aspartate aminotransferase (AST), Alanine
aminotransferase (ALT), Gamma-glutamyl-transferase, Cholesterol, Creatinine, Urea, Sodium, Potassium, Calcium, Chloride, Glucose, Bile acids, Albumin, Globulin, A/G ratio, Total protein
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during last week of exposure on 5 males and 5 females
- Dose groups that were examined: all
- Battery of functions tested: beam-walking and grip strength
(no separate test for motor activity see clinical signs))
IMMUNOLOGY: Yes
- Time schedule for examinations: from all adult males and high dose females (samples from other female dose groups were not analysed)
- Dose groups that were examined: all
- Parameters checked: T4 hormone (thyroxin) - Oestrous cyclicity (parental animals):
- Daily vaginal smears in all females fro 14 days before test start, during pre-mating and until successull mating
At termination a vaginal smear was taken to assess the estrous stage - Sperm parameters (parental animals):
- Histophatological examination of the testis was conducted with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure. This comprises identifying tubules with respect to their stage in the spermatogenic cycle for evaluation of germ cell death and depletion and spermatid retention
- Litter observations:
- STANDARDISATION OF LITTERS
: no
PARAMETERS EXAMINED
The following parameters were examined in offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD, day 4), presence of nipples/areolae in male pups (day 13)
GROSS EXAMINATION OF DEAD PUPS: yes for external abnormalities
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: yes blood sampling for thyroxine on day 4 and 13 from 2 pups/litter (only day 13 samples were analysed) - Postmortem examinations (parental animals):
- ORGAN WEIGHTS: Yes (see table)
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table) in addition the parathyroid and the skin of 5 animals/sex of the control and 1000/500 mg/kgbw group were investigated histopathologically. - Postmortem examinations (offspring):
- GROSS NECROPSY
- Gross necropsy consisted of external examinations
HISTOPATHOLOGY / ORGAN WEIGTHS
thyroid glands from one male and one female pup per litter were preserved (but not examined) - Statistics:
- The arithmetic mean and standard deviation were calculated for all grouped numerical data originating from monitoring the body weight, food- and water consumption and organ weights (gross pathology).
For basic analysis the respective test item groups were compared to the vehicle group by assessing of statistical significance using a two-tailed unpaired Student´s t-test. For all calculations, the significance level was set to 0,05.
In case the basic analysis returned a statistical significance additional inductive statistical analysis was applied as outlined in the main decision tree (see appendix). Except for individual blood parameters it was assumed that blood data collected in the present study were metrically scaled and normally distributed (Gaussian). Most statistical hypotheses in this study were best characterized as “many to one” – comparing a vehicle control vs. three treatment groups, respectively. Therefore the adequate analysis method was a One-Way ANOVA (Analysis of variance), followed by a post hoc Dunnett´s t-test (for organ weights), or two-way ANOVA followed by a post-hoc Bonferroni test (for body weight analysis). In case the One-Way ANOVA analysis detected a not normal distribution of residuals, the Kruskal-Wallis (H-test) analysis followed by a Dunn’s post hoc test was subsequently performed. For
all calculations, the significance level was set to 0,05. - Reproductive indices:
- none reported
- Offspring viability indices:
- none reported
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Salivation shortly after test item administration at 1000/500mg/kg bw and to a lesser extent at 250 mg/kg bw
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Males:
2 at 1000/500 mg/k bw: 1 gavage error on day 5 of pre-mating, 1 regurgitation from the stomach and aspiration on day 6 of mating
1at 250 mg/kg bw: 1 gavage error on day 3 of post-mating
Females
2 at 1000/500 mg/k bw: 1 gavage error on day 4 of pre-mating, 1 regurgitation from the stomach and aspiration on day 11 of mating
2 at 250 mg/kg bw: 1 gavage error on day 7 of -mating, 1 regurgitation from the stomach and aspiration on day 10 of pre-mating
The gavage related reflux was assumed to have a physicochemical relationship with the viscous test material rather than a toxicological property of the substance. - Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Regarding the body weight and the body weight gain, no significant differences were observed between all test item-treated animal groups (male and female) and their respective vehicle control groups. Occasional differences observed for the female animals at specific time intervals of the in life phase could be correlated with the animal’s pregnancy and lactation status and were therefore strongly assumed to be of natural origin
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- The monitored time intervals did not reveal any changes of toxicological relevance in regard to food consumption between the animal test groups and their respective vehicle control groups throughout male and female in-life phases. Male animals showed some differences after the mating procedure, which could be associated with the physical stress during the mating procedure. Some variances were observed for the female animal groups, which could be associated with the specific animal condition during late pregnancy.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- Male dose groups showed test item-related differences in water consumption, when compared to the respective vehicle control group. Some variances observed for the female animal groups, could be associated with the specific animal condition during phases of late pregnancy and subsequent lactation. Variations in water consumption during the complete in-life phase (males) could be related to the physical characteristics of the test item. However, a test item related effect cannot be excluded
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- Effects seen (see overview table) were all not significant and considered not related to treatment
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Males at 1000/500 mg/kg bw: sign decreased Albumin (-4%) and A/G ratio (-10%) within historical controls
Males at 250 mg/kg bw: sign decreased Albumin (-4%) within historical controls
Females at 1000/500 mg/kg bw: ALP sign decreased (-50%) and Potassium significantly increased (+19%) within historical controls
Other effects seen (see overview table) were all not significant and/or considered not related to treatment
No dose-resonse was observed - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- The grip-strength and beam-walking tests did not show abnormal findings
- Immunological findings:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Males and females at 1000/500 mg/kg bw: minimal to moderate mononuclear inflammatory cell infiltrates in the renal interstitium in 3/5 animals (not considered adverse)
No effects on the male and female reproductive organs. - Histopathological findings: neoplastic:
- not examined
- Description (incidence and severity):
- Analytical results (see attached table): concentrations measured were within 80-115% of nominal. The measured concentration on the 1000 mg/kg bw group fell outside the callibration range. The two additional samples at 500 mg/kg bw were within this range.
- Reproductive function: oestrous cycle:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The daily monitoring of the animal’s oestrus cycles revealed at least one regular cycle for each experimental female before mating
Irregular oestrus cycles were seen at an average frequency of 1/7, 0/6, 1/6 and 1/6 in females at 0, 62.5, 250 and 1000/500 mg/kg bw (expressed as mean irregular of total oestrus circles during pre-exposure (14 d), premating and mating period) - Reproductive function: sperm measures:
- no effects observed
- Description (incidence and severity):
- There were no test item-related microscopic observations in testes, epididymides, prostate gland, seminal vesicles or male mammary glands.
Germ cells in the testis were evaluated by identifying tubules with respect to their stage in the spermatogenic cycle. Neither germ cell depletion nor spermatid retention were found.
The population of Sertoli cells was normal and interstitial Leydig cells did neither show atrophy or hyperplasia. Epididymides did not show desquamated germ cells, cell debris or reduced number of sperm. - Reproductive performance:
- effects observed, treatment-related
- Description (incidence and severity):
- 1000/500mg/kg : 6 out of 11 animals achieved pregnancy after first mating; 4 animals achieved pregnancy after second mating
250 mg/kg bw: 9 out of 11 animals achieved pregnancy after first mating; No animal was included in the second mating; 2 animals (E256/0, E257/0) conceived on day 7
62.5 mg/kg: 11 out of 12 animals achieved pregnancy after first mating; No animal was included in the second mating
Vehicle: 10 out of 12 animals achieved pregnancy after first mating; No animal was included in the second mating
Duration of the first mating period up to 14 days.
The available data do not give any indication if the initial high dose of 1000 mg/kg during the first 28 days (i.e. also during the first mating period) could be responsible for the reduced number of pregnant females, and whether the reduction to 500 mg/kg was finally decisive for the sufficient number of pregnant females after the second mating period. - Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day
- Based on:
- act. ingr.
- Sex:
- male/female
- Basis for effect level:
- reproductive performance
- Remarks on result:
- other: a final conclusion could not be drawn (worst case approach)
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 500 mg/kg bw/day (nominal)
- System:
- other: fertility could be affected at 1000/500 mg/kg bw
- Organ:
- other: no effects on any organ system, a final conclusion cannot be drawn
- Treatment related:
- yes
- Dose response relationship:
- yes
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No difference in pup development could be identified between the pups of the dose groups and the pups of the vehicle group. All pups from substance-treated dams showed a regular physical appearance, no substance-related differences in physical development were identified between dose group pups and vehicle control group pups during the course of the study.
- Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- The increased mortality rate (+2.2%) in 1000/500 mg/kg bw litters was attributed to mortality in one litter (see attachment)
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- The decreased body weight (gain) at 1000/500 mg/kgbw was attributed to one litter with only 2 pups (see attachment)
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- no gross abnormalities related to the administration of the substance were detected
- Description (incidence and severity):
- The sex ratio (expressed as percentage males) was 48, 53, 45 and 51% at 0, 62.5,250 and 1000/500 mg/kg bw (see table)
No effects on nipple reterntion were observed (see attachment)
The anogenital distance in males and females did not differ between treatment groups and controls (see table) - Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- no effects observed
- Description (incidence and severity):
- no substance-related changes in total T4-hormone content in blood plasma could be detected (see attachment)
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 500 mg/kg bw/day
- Based on:
- act. ingr.
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects observed
- Critical effects observed:
- no
- Reproductive effects observed:
- yes
- Lowest effective dose / conc.:
- 500 mg/kg bw/day
- Treatment related:
- yes
- Relation to other toxic effects:
- reproductive effects as a secondary non-specific consequence of other toxic effects
- Dose response relationship:
- yes
- Conclusions:
- Based on the information in the study report the NOAEL for reproduction is 250 mg/kg bw. The NOAEL for developmental effects is 1000/500 mg/kg bw.
- Executive summary:
The substance was administered daily by oral gavage to 12 male and female adult rats in three groups at dosages of 500 mg (1000 mg during study days 1-28), 250 mg and 62,5 mg test item per kg body weight over a period of at least 28 days (males) and at least 49 days (females). Male and female animals were mated within their treatment group during the treatment period. Litters were used for evaluation of developmental and reproduction related parameters but did not receive test item treatment. Further 24 animals (12 males and 12 females) received the vehicle solution as control; all other parameters were identical.
Clinical signs and viability were monitored daily during the in-life phase. Individual body weight was recorded once weekly. Individual food / water consumption of female animals and group food / water consumption of male animals were recorded once weekly. Detailed clinical signs were monitored weekly from all adult animals. Grip strength and reactivity to stimuli (beam-walking test) was assessed from 5 randomly selected adult animals per sex and group during the last exposure week.
At the end of the treatment period, blood samples were taken from all adult animals to provide T4-hormone data. For assessment of additional toxicological parameters, 5 adult animals per sex and group were randomly selected for additional blood sampling (females during end of pre-mating, males at day of necropsy) to provide data on hematology and clinical biochemistry.
All adult and offspring animals were sacrificed after bleeding and examined by gross necropsy. Weights of selected organs were recorded and selected tissues and organs were preserved with special emphasis on organs of the reproductive system. From 5 randomly selected adult animals per sex and group, additional organs were preserved and examined microscopically.
Offspring parameters were assessed from each litter on day 0 post partum (pp), day 4pp, day 13pp and day of necropsy as specified by the study plan. These included numbers, stillbirth, live birth and runts, mortality, sex and presence of gross abnormalities. Blood samples from selected offspring per litter were taken on day 4pp to provide T4-hormone data.
The results of the repeated dose part can be found under repeated dose toxicity.
Regarding reproduction and development (achievement and duration of pregnancy, offspring losses, survival rate of pups and development of pups) no difference was observed for all test item treated-animals, when compared to the vehicle control animals. There was no evidence for any test item-related effect.
Conclusion:
A daily oral administration of the substance to male and female Wistar rats at dose levels of 500 mg (1000 mg during study days 1-28), 250 mg and 62,5 mg/kg body weight over a time period of 47-72 days for males and 51-91 days for females resulted in some minor animal behavioral changes observed in male and female animals predominately of the high dose groups, alterations in water consumption in male dose groups, changes in clinical biochemistry (AP lower and K higher) in the female high dose group and microscopic changes in the kidneys of male and female high dose animals. The findings were not considered adverse. As the findings could not be associated with a substantial impact on the animal’s health, the NOAEL regarding the repeated dose toxicity was set to 250 mg/kg body weight.
Furthermore, no pathological evidence for toxic effects on the reproduction performance of female and male rats was found. Regarding the overall time period of gestation/lactation and after birth until end of in-life phase no evidence for a toxic effect on the pup development could be detected. The NOAEL regarding reproduction and development of the substance under these study conditions was set to be 250 mg/kg body weight for the female and male animals.
Reference
There were no test item-related microscopic observations in testes, epididymides, prostate gland, seminal vesicles or male mammary glands. There were no test item-related microscopic observations in ovaries, uterus, cervix or vagina.
For all female animals examined ovaries showed numerous large eosinophilic corpora lutea, typical of postparturition. Most animals had undergone a post parturition estrus cycle and were in the late metestrus, diestrus, or early proestrus phase. There was general concordance between the estrus cycle determined on vaginal mucosa by histopathology and the estrus cycle determined on vaginal smears.
There were no effects on sex ratio, thyroid hormone levels, nipple retention and anogenital distance.
Dose |
0 |
|
62.5 |
|
250 |
|
1000/500 |
|
Treatment related |
Endpoint |
M |
F |
M |
F |
M |
F |
M |
F |
|
Litter size day 1 (day 13) |
10.4 (9.6) |
11.4 (10.1) |
9.7 (8.9) |
9.0 (8.3*) |
no |
||||
BW litter (day 1/13) |
63.8/296 |
72.2/316 |
60.4/268 |
58.3/258* |
no |
||||
Pup weight (day 1/13) |
6.1/31.2 |
6.4/31.5 |
6.4/31.4 |
6.6/29.2* |
no |
||||
Pup mortality (%) day 0-4 Day 4-14 |
0.0 0.0 |
1.6 0.0 |
0.0 0.0 |
2.2# 0.0 |
no |
||||
Sex ratio (% males) |
48 |
53 |
45 |
51 |
no |
||||
Thyroxine day 13 (nmol/L) |
93.3 |
92.6 |
88.8 |
91.8 |
no |
||||
Anogenital distance (normalized mm) |
2.2 |
1.3 |
2.2 |
1.3 |
2.1 |
1.2 |
2.2 |
1.3 |
no |
Nipple count |
NTRE |
no |
|||||||
Macroscopy |
NTRE |
no |
↑/↓= significantly increased/decreased
NTRE no treatment related effects
*Decrease compared to controls can be attributed to one litter
# Increase compared to controls can be attributed to one litter
Effect on fertility: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 250 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- no definitive conclusion can be drawn for the effects observed in the high dose groups, therefore a worst case approach has been taken
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
It is uncertain whether the effects on mating performance during the first 28 days of the study at the 1000 mg/kg bw group can be related to the substance, although it seems that the reduction of the dose to 500 mg/kg bw has influence on the success of the second mating. No definitive conclusion can be drawn related to the influence of other toxic effects of the substance during this first 28 days of the study and whether the effect on mating performance seen is secundary to these effects.
Effects on developmental toxicity
Description of key information
In a test according to OECD 422 the substance was administered daily by oral gavage to 12 male and female adult rats in three groups at dosages of 500 mg (1000 mg during study days 1-28), 250 mg and 62,5 mg test item per kg body weight over a period of at least 28 days (males) and at least 49 days (females). Male and female animals were mated within their treatment group during the treatment period. Litters were used for evaluation of developmental and reproduction related parameters but did not receive test item treatment. Further 24 animals (12 males and 12 females) received the vehicle solution as control; all other parameters were identical.
Clinical signs and viability were monitored daily during the in-life phase. Individual body weight was recorded once weekly. Individual food / water consumption of female animals and group food / water consumption of male animals were recorded once weekly. Detailed clinical signs were monitored weekly from all adult animals. Grip strength and reactivity to stimuli (beam-walking test) was assessed from 5 randomly selected adult animals per sex and group during the last exposure week.
At the end of the treatment period, blood samples were taken from all adult animals to provide T4-hormone data. For assessment of additional toxicological parameters, 5 adult animals per sex and group were randomly selected for additional blood sampling (females during end of pre-mating, males at day of necropsy) to provide data on hematology and clinical biochemistry.
All adult and offspring animals were sacrificed after bleeding and examined by gross necropsy. Weights of selected organs were recorded and selected tissues and organs were preserved with special emphasis on organs of the reproductive system. From 5 randomly selected adult animals per sex and group, additional organs were preserved and examined microscopically.
Offspring parameters were assessed from each litter on day 0 post partum (pp), day 4pp, day 13pp and day of necropsy as specified by the study plan. These included numbers, stillbirth, live birth and runts, mortality, sex and presence of gross abnormalities. Blood samples from selected offspring per litter were taken on day 4pp to provide T4-hormone data.
The results of the repeated dose part can be found under repeated dose toxicity.
Regarding reproduction and development (achievement and duration of pregnancy, offspring losses, survival rate of pups and development of pups) no difference was observed for all test item treated-animals, when compared to the vehicle control animals. There was no evidence for any test item-related effect.
Conclusion:
A daily oral administration of the substance to male and female Wistar rats at dose levels of 500 mg (1000 mg during study days 1-28), 250 mg and 62,5 mg/kg body weight over a time period of 47-72 days for males and 51-91 days for females resulted in some minor animal behavioral changes observed in male and female animals predominately of the high dose groups, alterations in water consumption in male dose groups, changes in clinical biochemistry (AP lower and K higher) in the female high dose group and microscopic changes in the kidneys of male and female high dose animals. The findings were not considered adverse. As the findings could not be associated with a substantial impact on the animal’s health, the NOAEL regarding the repeated dose toxicity was set to 250 mg/kg body weight.
Furthermore, no pathological evidence for toxic effects on the reproduction performance of female and male rats was found. Regarding the overall time period of gestation/lactation and after birth until end of in-life phase no evidence for a toxic effect on the pup development could be detected. The NOAEL regarding reproduction and development of the substance under these study conditions was set to be 250 mg/kg body weight for the female and male animals.
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 500 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
Based on the information available, classification of the substance according to Regulation (EC) No 1272/2008 (CLP) for reproduction and developmental toxicity is not considered necessary.
Additional information
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