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EC number: 917-830-2 | CAS number: 1186514-91-9
- 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
Repeated dose toxicity: oral
Administrative data
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016-2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Guideline study under GLP
Cross-reference
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016=2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Guideline study under GLP
- Reason / purpose for cross-reference:
- reference to same 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
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- - Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation:369-460 g (males); 225-288 (females)
- Fasting period before study: no, fasting only before sacrifice
- Housing: males housed in groups of 4, in polycarbonate cages. Females housed singly during and after mating. Lignocell (R) and Arbocel (R) natural crinklets
- Diet (e.g. ad libitum): at lib, ssniff (R)
- Water (e.g. ad libitum): ad lib, municipal water supply
- Acclimation period: at least 5 days
DETAILS OF FOOD AND WATER QUALITY: ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/3
- Humidity (%): 30-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 29 Nov 2016 To: 13 Jan 2017. - Route of administration:
- oral: gavage
- Vehicle:
- olive oil
- Remarks:
- PEG 400
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water): appropriate vehicle for oily liquid test material
- Concentration in vehicle: 100, 300 and 1000 mg/kg bw/d
- Amount of vehicle (if gavage): stability measured at concentrations up to 250 mg/ml at room temperature.
- Lot/batch no. (if required): BCBQ0052V, Sigma-Aldrich
- Purity: - Details on mating procedure:
- - M/F ratio per cage: 1/1
- Length of cohabitation: until copulation or up to 14 days
- Proof of pregnancy:vaginal plug or sperm in vaginal smear/ referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: yes, for up to 7 days
- After successful mating each pregnant female was caged individually
- Any other deviations from standard protocol: - Analytical verification of doses or concentrations:
- yes
- Remarks:
- See Analytical Report FPBSTUDY-156-VAL1: Torok J, Validation of Analytical Method for the Determination of ZEF 6099/100 from Gavage Media. FumoPrep Ltd., H-1044 Budapest, Ipari park u. 10, Hungary.
- Details on analytical verification of doses or concentrations:
- Instrument: Near infrared spectrophotomer: MicroNIR™ Pro 1700 ES
Mode: Transmission
Range: 908 – 1676 nm
Integration time: 10.2 ms
Scan Count: 100
Cuvette: Quartz, 1.000 cm
Objective: multivariate evaluation 5-5 NIR spectra of the test item solution at 4.8 mg/mL concentration level. The spectra of the test solution and the vehicle were compared. The spectra were pre-treated with standard normal variate normalization. There was intensive signal of the vehicle. The principal component analysis of the spectral data matrix eliminated the variables that carried no variance, resulting in spectra are informative.
Sample size: 7
LOQ: 4.90 mg/mL (0.542 m/m%)
Recovery:
92.3% at 15 mg/ml test solution.
91.4 % at 60 mg/ml
106.1% at 200 mg/ml
Precision:
5.79 % at 15 mg/ml test solution.
1.21 % at 60 mg/ml
1.06% at 200 mg/ml
The accuracy of the calibration solution was between 98.5 and 102.6% with 2.32% RSD (n=3) - Duration of treatment / exposure:
- 28 days (males), up to 44 days (females)
- Frequency of treatment:
- daily
- Details on study schedule:
- Dosing of Male Animals:
Acclimatisation At least 5 days
Pre-mating period 14 days
Mating/Post-mating period at least 14 days
Last week of treatment: FOB (e.g. Day 25 am, 5 animals/group)
Prior to/at necropsy: Urinalysis, i.e. sampling start on Day 27, end Day 28 after approximately 16 hours (5 animals/group). Coagulation evaluation, Clinical chemistry and Haematology (5 animals/group). Necropsy with organ weight determination (i.e. Day 28 or later, all males).
Females:
Acclimatisation period: at least 5 days
Pre-mating period: 14 days
Mating: Up to 14 days
Gestation: 22-24 days
Delivery: 1 day
Lactation period: at least 4 days: FOB (5 animals/group) on PND4, Pups necropsy on PND 4. Dams fasted overnight prior to necropsy.
PND5: Prior to/at necropsy:
- Urinalysis (i.e., sampling start on PND 4, end PND5 after approximately 16 h, 5 animals/group).
- Coagulation evaluation, Clinical chemistry and Haematology (5 animals/group)
- Necropsy with organ weight determination (i.e. all females)
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 12/sex/group
- Control animals:
- yes, concurrent vehicle
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to first dose, and at least weekly
BODY WEIGHT: Yes
- Time schedule for examinations: at randomisation, before treatment, at day 0, weekly and at necr opsy. Females also at GD0, 3, 10, 17 and 20 and at PPD 0 and 4.
FOOD CONSUMPTION AND COMPOUND INTAKE: not a feeding study
WATER CONSUMPTION AND COMPOUND INTAKE: not a drinking water study
OTHER:
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:
HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (pentobarital)
- Animals fasted: Yes, prior to sacrifice
- How many animals:
- Parameters checked in table [No.?] were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals:
- Parameters checked in table [No.?] were examined.
URINALYSIS: Yes
- Time schedule for collection of urine: Urine sampling will be performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.?] were examined.
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.
OTHER:
Sacrifice and pathology
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, on the control and high dose group animals.
Other examinations
Functional Observation Battery on 5 animals/group.
Body and organ weight: At the time of termination, body weight and weight of the following organs of all adult animals will be determined:
uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus, adrenals, ovaries, thyroids with parathyroids. Testes and epididymides will be weighed individually. Individual and/or paired absolute organ weight will be reported for each animal and adjusted for the body and brain weights. Paired organ weights as ap plicable will be summarised. Relative organ weight (to body and brain weight) will be calculated and reported. - Oestrous cyclicity (parental animals):
- - Number of pairings
- Number of pregnant females
- Number of sperm positive, but non-pregnant females
- Number of non mated females
- Duration of pregnancy (days)
- Number of Corpora lutea / dams
- Number of implantations / dams
- Number of dams with live pups Day 0 and 4
- * Pre-implantation mortality
- * Intrauterine mortality
- * Total mortality (intra and extra uterine mortality)
Detailed histological examination of the ovaries will cover the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. - Sperm parameters (parental animals):
- - Number of pairings
- Number of fertile pairings
- Number of infertile males
Special attention will be paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. - Litter observations:
- Females will be allowed to litter and rear their offspring. Delivery process will be observed as carefully as possible. Dams will be observed to record whether they form a nest from the bedding material and cover their new-borns or not. The efficiency of suckling will be observed by the presence of milk in the pups' stomach. Each litter will be examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), the presence of gross abnormalities and any abnormal behaviour of the offspring. Live pups will be counted, sexed, weighed individually within 24 hours of parturition (ex. Day 0 or 1 post-partum, PND0 or 1) and on PND4, with accuracy of 0.01g. All the litters will be checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) will be subjected to necropsy with macroscopic examination and the cause of death will be identified if possible.
- Postmortem examinations (parental animals):
- Gross necropsy will be performed on each animal. Terminally (one day after the last treatment), animals will be sacrificed under anaesthesia by exsanguination; anaesthetic product may be diluted for pups’ euthanasia as required.
For the adult animals, detailed histological examination will be performed as follows:
• on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group),
• any animals found dead or euthanized pre-terminally during the study in all groups
• all macroscopic findings (abnormalities), except of minor order from all animals
• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups and of all males that failed to sire and all females that failed to deliver healthy pups - Postmortem examinations (offspring):
- - Mean pup body weight (per pup within the group and per litter) on PND 0 and 4
- Mean pup body weight gain (per litter) between postnatal Days 0-4
The pups found dead and intact (not cannibalized) will be subjected to necropsy with macroscopic examination and the cause of death will be identified if possible. All observed abnormalities will be recorded. - Statistics:
- Data is collected using software PROVANTIS v.9. Statistical analysis is performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS v9.2 (when using Provantis).
In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups will be checked by Bartlett's test. Where no significant heterogeneity is detected, a one-way analysis of vari ance (ANOVA) is carried out. If the obtained result is significant, then Duncan's Multiple Range test is used to assess the significance of inter-group differences. Where significant heterogeneity is found, the normal distribution of data is examined by Kolmogorow-Smirnow test. In the case of non- normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance is applied. If a positive result is detected, the inter-group comparisons are performed using Mann-Whitney U-test. The Chi-squared test will be used for non-continuous data.
In case of the SAS 9.2 software package (within the validated Provantis system) the following decision tree is applied automatically for statistical evaluation of continuous numeric data. The normality and heterogeneity of variance between groups will be checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log- transformed when justified). Where both tests show no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test is carried out. If the obtained result is positive, Dunnett (Multiple Range) test is used to assess the significance of inter- group differences; identifying differences of <0.05 or <0.01 as appropriate. - Reproductive indices:
- * Female mating index
* Female fertility index
* Gestation index
* Male mating index
* Male fertility index - Offspring viability indices:
- * Survival Index of pups on postnatal Days 0 and 4
* Sex ratio % (on postnatal Days 0 and 4)
Number of live births per litter, and number of viable pups per litter on postnatal Days 0 and 4 - Clinical signs:
- no effects observed
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- Low dose males showed decreased basophillic neutrophil concentrations. Not treatment related/toxicologically significant
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- Low dose males showed decreased protein concentrations. Not treatment related/toxicologically significant
- Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- Low dose males showed altered urinary pH. Not treatment related/toxicologically significant
- Behaviour (functional findings):
- no effects observed
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- not examined
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- Mating and fertility indices were 100% for both males and females. Successful matings occurred within 3 days for all dose groups. The gestation index was 100% for all groups other than low dose females, which showed a gestation index of 92%.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- > 1 000 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- clinical signs
- body weight and weight gain
- food consumption and compound intake
- haematology
- clinical biochemistry
- urinalysis
- organ weights and organ / body weight ratios
- gross pathology
- histopathology: non-neoplastic
- reproductive function (oestrous cycle)
- reproductive function (sperm measures)
- reproductive performance
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Remarks:
- NOAEL > 1000 mg/kg bw/d
- Key result
- Critical effects observed:
- no
- Clinical signs:
- no effects observed
- Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- There were 3 Control, 1 Low Dose, 4 Mid Dose and 1 High dose pups found dead. The cannibalization of 1, 2, 4 and 1 pups from the Control, Low, Mid and High dose groups respectively was observed at necropsy.
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- not examined
- Description (incidence and severity):
- No test item-related macroscopic findings were observed in the F1 generation in any dose group (PN0-4).
- Histopathological findings:
- not examined
- Other effects:
- not examined
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- > 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- viability
- clinical signs
- mortality
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Remarks:
- The NOAEL is greater than 1000 mg/kg bw/d.
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- no
- Lowest effective dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Treatment related:
- no
- Conclusions:
- In an OECD 422 guideline study on ZEF 6099/100 at doses of 100, 300 and 1000 mg/kg bw/d in Wistar rats, no adverse effects were observed in fertility, mating, gestation or development of the offspring. No teratological effects were observed. The NOAEL is greater than 1000 mg/kg bw/d.
The sex ratio of female pups was lower (not statistically significant) in all test item treated groups (both on PND0 and PND4) when compared to the control data, therefore it was not considered as a test item effect.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
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)
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- CiToxLab Hungary Ltd. H-8200 Veszprém, Szabadságpuszta, Hungary
- Limit test:
- no
Test material
- Reference substance name:
- {3-hydroxy-4-[(9Z)-octadec-9-enoyloxy]cyclohexyl}methyl 3-hydroxy-4-[(9Z,12Z)-octadeca-9,12-dienoyloxy]cyclohexane-1-carboxylate; {3-hydroxy-4-[(9Z,12Z)-octadeca-9,12-dienoyloxy]cyclohexyl}methyl 3-hydroxy-4-[(9Z,12Z)-octadeca-9,12-dienoyloxy]cyclohexane-1-carboxylate
- EC Number:
- 917-830-2
- Cas Number:
- 1186514-91-9
- Molecular formula:
- not available for UVCB substances
- IUPAC Name:
- {3-hydroxy-4-[(9Z)-octadec-9-enoyloxy]cyclohexyl}methyl 3-hydroxy-4-[(9Z,12Z)-octadeca-9,12-dienoyloxy]cyclohexane-1-carboxylate; {3-hydroxy-4-[(9Z,12Z)-octadeca-9,12-dienoyloxy]cyclohexyl}methyl 3-hydroxy-4-[(9Z,12Z)-octadeca-9,12-dienoyloxy]cyclohexane-1-carboxylate
- Test material form:
- liquid
- Details on test material:
- Lot # 210164, brown liquid, homogeneous, Expiry date: 2017-12-16
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 Services, Germany GmbH
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation:
- Fasting period before study: no, fasting only before sacrifice
- Housing: males housed in groups of 4, in polycarbonate cages. Females housed singly during and after mating. Lignocell (R) and Arbocel (R) natural crinklets
- Diet (e.g. ad libitum): at lib, ssniff (R)
- Water (e.g. ad libitum): ad lib, municipal water supply
- Acclimation period: at least 5 days
DETAILS OF FOOD AND WATER QUALITY:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/3
- Humidity (%): 30-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: To:
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- olive oil
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water): appropriate vehicle for oily liquid test material
- Concentration in vehicle: 100, 300 and 1000 mg/kg bw/d
- Amount of vehicle (if gavage): stability measured at concentrations up to 250 mg/ml at room temperature.
- Lot/batch no. (if required):
- Purity: - Analytical verification of doses or concentrations:
- yes
- Remarks:
- See Analytical Report FPBSTUDY-156-VAL1: Torok J, Validation of Analytical Method for the Determination of ZEF 6099/100 from Gavage Media. FumoPrep Ltd., H-1044 Budapest, Ipari park u. 10, Hungary.
- Details on analytical verification of doses or concentrations:
- Instrument: Near infrared spectrophotomer: MicroNIR™ Pro 1700 ES
Mode: Transmission
Range: 908 – 1676 nm
Integration time: 10.2 ms
Scan Count: 100
Cuvette: Quartz, 1.000 cm
Objective: multivariate evaluation 5-5 NIR spectra of the test item solution at 4.8 mg/mL concentration level. The spectra of the test solution and the vehicle were compared. The spectra were pre-treated with standard normal variate normalization. There was intensive signal of the vehicle. The principal component analysis of the spectral data matrix eliminated the variables that carried no variance, resulting in spectra are informative.
Sample size: 7
LOQ: 4.90 mg/mL (0.542 m/m%)
Recovery:
92.3% at 15 mg/ml test solution.
91.4 % at 60 mg/ml
106.1% at 200 mg/ml
Precision:
5.79 % at 15 mg/ml test solution.
1.21 % at 60 mg/ml
1.06% at 200 mg/ml
The accuracy of the calibration solution was between 98.5 and 102.6% with 2.32% RSD (n=3) - Duration of treatment / exposure:
- 28 days for males, up to 58 days for females
- Frequency of treatment:
- once daily, 7 days per week.
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:
- 12/sex/group
- Control animals:
- yes, concurrent vehicle
Examinations
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked in table [No.?] were included.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to first dose, and at least weekly
BODY WEIGHT: Yes
- Time schedule for examinations: at randomisation, before treatment, at day 0, weekly and at necropsy. Females also at GD0, 3, 10, 17 and 20 and at PPD 0 and 4.
OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (pentobarital)
- Animals fasted: Yes, prior to sacrifice
- How many animals:
- Parameters checked in table [No.?] were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals:
- Parameters checked in table [No.?] were examined.
URINALYSIS: Yes
- Time schedule for collection of urine: Urine sampling will be performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.?] were examined.
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.
OTHER: - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, on the control and high dose group animals. - Other examinations:
- Functional Observation Battery on 5 animals/group.
Body and organ weight: At the time of termination, body weight and weight of the following organs of all adult animals will be determined:
uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus, adrenals, ovaries, thyroids with parathyroids. Testes and epididymides will be weighed individually. Individual and/or paired absolute organ weight will be reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable will be summarised. Relative organ weight (to body and brain weight) will be calculated and reported.
The number of implantation sites and of corpora lutea will be recorded in the females as applicable. - Statistics:
- Data is collected using software PROVANTIS v.9. Statistical analysis is performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS v9.2 (when using Provantis).
In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups will be checked by Bartlett's test. Where no significant heterogeneity is detected, a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant, then Duncan's Multiple Range test is used to assess the significance of inter-group differences. Where significant heterogeneity is found, the normal distribution of data is examined by Kolmogorow-Smirnow test. In the case of non- normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance is applied. If a positive result is detected, the inter-group comparisons are performed using Mann-Whitney U-test. The Chi-squared test will be used for non-continuous data.
In case of the SAS 9.2 software package (within the validated Provantis system) the following decision tree is applied automatically for statistical evaluation of continuous numeric data. The normality and heterogeneity of variance between groups will be checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log- transformed when justified). Where both tests show no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test is carried out. If the obtained result is positive, Dunnett (Multiple Range) test is used to assess the significance of inter- group differences; identifying differences of <0.05 or <0.01 as appropriate.
Results and discussion
Results of examinations
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No clinical signs were noted which were associated with the test material. Four males in the low dose group displays topical scars (with or without wounds) due to fighting. Red eye discharge was observed in one high dose male. No signs were noted in female animals.
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- A statisitically significant decrease in the amount of basophilic neutrophils was observed in high dose males, but this was within the normal historical range of values. There was no similar decrease in the high dose females. Therefore, this was not considered toxicologically significant.
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- A statisitically significant decrease in total protein concentration was observed in low dose males, but the data from all males was within the normal historical range of values, and there was no dose response. It is not considered toxicologically relevant or related to treatment.
- Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- Statisitically significant differences in pH of the urine were observed in low and mid dose males, but this was within the normal historical range of values; it was not dose related, and did not occur in females. It was considered unrelated to treatment.
- Behaviour (functional findings):
- no effects observed
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Description (incidence and severity):
- Discussed as behavioural findings using the Functional Observational Battery.
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- Several findings typical of background conditions found in this strain of rat were observed and considered incidental. This included: minimal cardiomyocytes degeneration in 2/5 males and 1/5 females, minimal mixed mononuclear infiltrate in the myocardium of 1/5 males, slight tubular degeneration of right kidney in 1/5 males, minimal tubular basophilia of right kidney in 1/5 males and minimal intraluminal mineralization of the right kidney (papilla) in 1/5 males, minimal focal hepatocellular necrosis in 1/5 males, and minimal multinucleated giant cells in the testes of 1/5 males.
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Details on results:
- No test material related changes were noted in any parameter examined in this study.
Effect levels
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- > 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Remarks:
- NOAEL > 1000 mg/kg bw/d
Target system / organ toxicity
- Key result
- Critical effects observed:
- no
Applicant's summary and conclusion
- Conclusions:
- In an OECD 422 guideline study of ZEF 6099/100 in the Wistar rat at doses of 100, 300 and 1000 mg/kg bw/d, the test material did not result in adverse effects in clinical signs, body weight, organ weights, food consumption, neurological assessment, haematology, coagulation, clinical chemistry, urinalysis, or gross/histopathology. There were no effects in reproductive parameters nor in F1 offspring viability, clinical signs, development or macroscopic examination. The NOAEL is greater than 1000 mg/kg bw/d.
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