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EC number: 613-848-7 | CAS number: 65870-94-2
- 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
Developmental toxicity / teratogenicity
Administrative data
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 20 February 2015 to 04 December 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study
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:
- other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- yes
- Remarks:
- for 1 female in group 1, bodyweight and food consumption were recorded on Day 1 p.c.Proof of mating on the first day of estrous evaluation (Day 0 p.c.). Not thought to impact the integrity of the results
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
Test material
- Reference substance name:
- 2-ethyl-2-(((3,5,5-trimethylhexanoyl)oxy)methyl)propane-1,3-diyl bis(3,5,5-trimethylhexanoate)
- EC Number:
- 613-848-7
- Cas Number:
- 65870-94-2
- Molecular formula:
- C33H62O6
- IUPAC Name:
- 2-ethyl-2-(((3,5,5-trimethylhexanoyl)oxy)methyl)propane-1,3-diyl bis(3,5,5-trimethylhexanoate)
- Details on test material:
- - Physical state: Clear viscous fluid
- Storage condition of test material: Room temperature protected from light
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- A total of 124 Sprague-Dawley RjHan:SD rats (62 males and 62 females) were received on 03 March 2015.
- On the first day of treatment, males were approximately 10 weeks old and females were approximately 9 weeks old.
- At the start of treatment, mean body weight was 422 g (range: 375 g to 458 g) for males and 255 g (range: 224 g to 277 g).
- Males and females were sexually mature and were not siblings.
- Females were virgin.
- Animals were clinically examined on arrival to ensure they were in good health.
- The animals were acclimated to the study conditions for a period of 8 days before the beginning of the treatment period. A larger number of animals than necessary were acclimated to permit selection and/or replacement of individuals.
- During the acclimation period, the required number of animals (60 males and 60 females) was selected according to body weight and clinical condition. The animals were allocated to groups (by sex) using a computerised randomisation procedure based on body weight so that the average body weight of each group was similar.
- Each animal was identified by an individual ear tattoo or on the tail. At the beginning of the study, each animal received a unique identity number.
ENVIRONMENTAL CONDITIONS
- Animals were housed in a barriered rodent unit from the point of receipt.
- Temperature: 22 ± 2 °C
- Relative humidity: 50 ± 20 %
- Lighting: 12 hours light and 12 hours dark
- Ventilation: 8 to 15 changes/hour of filtered, non-recylced, air
- The corresponding instrumentation and equipemtn were checked and calibrated at regular intervals.
- Temperature and relative humidity were recorded continuously (recording devices equipped with alarm systems).
- The animal room was disinfected before the arrival of the animals and cleaned regularly thereafter.
HOUSING
- Except during pairing and lactation, animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen in order to not jeopardise gestations and to avoid aggressive behaviour around mating between males
- Towards the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were placed in each cage of females.
- Each cage contained an object (rat hut) to provide environmental enrichment.
- The cages were placed in numerical order on the racks.
FOOD AND WATER
- All animals had free access to SSNIFF R/M-H pelleted maintenance diet (batch 4482794; SSNIFF Spezialdiaten GmbH, soest, Germany), which was distributed weekly.
- Animals had free access to bottles containing tap water (filtered with a 0.22 µm filter).
CONTAMINANT ANALYSES
- Batches of diet, sawdust and wood shaving were analysed by the suppliers for composition and contaminant levels.
- Bacterial and chemical analyses of water were performed regularly by external laboratories. These analyses included the detection of possible contaminants (pesticides and heavy metals).
- No contaminants were present in the diet, drinking water, sawdust or wood shavings at levels which could be expected to interfere with or prejudice the the outcome of the study.
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Remarks:
- (Batch number MKBQ9948V)
- Details on exposure:
- DURATION
- Dose formulations were administered to males daily for 2 weeks before mating, during the mating period and until sacrifice or treatment-free period (at least 5 weeks of treatment in total).
- Dose formulations were administered to females for 2 weeks before mating, during the mating period, during gestation, during lactation until day 5 post-partum inclusive or until sacrifice for females with no delivery.
- At the end of the treatment period, the animals were sacrificed expect for certain animals in groups 1 and 5 (the last five surviving males and the first five surviving lactating females). Animals that were not sacrificed were kept for a 2 week treatment-free period.
ADMINISTRATION
- Dose formulations were administered once daily, at approximately the same time of day, by gavage using a plastic syringe fitted with a metal gavage tube.
- The quantity of dose formulationadministered to each animal was adjusted according to the most recently recorded body weight.
- A constant dose volume of 4 mL/kg bw/day was used.
- Control animals (group 1) received the vehicle alone.
- The control dose formulation was stirred just before administration and the test item dose formulations for 15 minutes before administration.
- Formulations were maintained under continuous magnetic stirring throughout the dosing procedure. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - Gas Chromatography with FID detection (GC-FID)
- Analytical method provided by the sponsor and validated prior to dose formulation analysis.
- Test item concentration in dose formulations was determined once on formulations used in weeks 1, 3 and 6. A sample was taken from control and test item dose formulations and analysed using the validated method.
- Measured test item concentration was found to equal nominal concentration ± 10 %. - Details on mating procedure:
- MATING
- Females were paired with males from the same dose-level group.
- One female was placed with one male in the latter's cage during the night.
- Confirmation of mating was made in the morning by checking for the presence of a vaginal plug or for sperm in a vaginal lavage.
- The day of confirmed mating was designated day 0 post-coitum.
- Each female was placed with the same male until mating occurred.
- The pre-coital time was calculated for each female.
PARTRUITION
- Females were allowed to litter normally and rear their progeny until day 5 post-partum.
- Any sign of a difficult or prolonged partruition was recorded.
- The morning when partruition was completed was designated day 1 post-partum.
- The length of gestation was calculated. - Duration of treatment / exposure:
- - Males: 2 weeks before mating, during the mating period, then until sacrifice or treatment-free period (at least 5 weeks)
- Females: 2 weeks before mating, during gestation, during lactation until day 5 post partum (inclusive). Females that didn't deliver were dosed until sacrifice. - Frequency of treatment:
- Once daily
- Duration of test:
- 61 days
Doses / concentrationsopen allclose all
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- - Group 1: 0 mg/kg bw/day (15 males including 5 treatment free males and 15 females including 5 treatment-free females )
- Group 2: 100 mg/kg bw/day (10 males and 10 females)
- Group 3: 250 mg/kg bw/day (10 males and 10 females)
- Group 4: 500 mg/kg bw/day (10 males and 10 females)
- Group 5: 1000 mg/kg bw/day (15 males including 5 treatment free males and 15 females including 5 treatment-free females ) - Control animals:
- yes, concurrent no treatment
- yes, concurrent vehicle
- Details on study design:
- ESTROUS CYCLICITY (Parental animals)
- The estrous cyclce stage was determined from a fresh vaginal lavage (stained with methylene blue).
- The procedure was repeated each morning during the mating period until the females were mated.
SPERM PARAMETERS (Parental animals)
- During microscopic examination following sacrifice, special emphasis was placed on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Examinations
- Maternal examinations:
- CLINICAL EXAMINATIONS (F0 ANIMALS)
Morbidity and mortality: Each animal was checked for mortality or signs of morbidity once a day before the treatment period and at least twice a day during the treatment and treatment-free periods (including weekends and public holidays).
Clinical signs: From arrival, each animal was observed once a day as part of routine examinations. From the start of the treatment period, each animal
was observed once a day, at approximately the same time each day, for the recording of clinical signs.
MEDICAL CARE
- Cothivet, an antiseptic and healing product, was applied on lesions of animals D27526 (group 1), D27474 (group 3) and D27477 (group 4) for 2 or 5 days.
BODY WEIGHT
- The body weight of each male was recorded on the first day of treatment (day 1) and then once per week until sacrifice.
- The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mated, on days 0, 7, 14 and 20 post-coitum, and on days 1 and 5 post-partum. Body weights of treatment-free females were recorded on days 12 and 19.
FOOD CONSUMPTION
- The quantity of food consumed by each male was measured once a week over a 7 day period from the first day of treatment until the start of the mating period, and then once a week during the treatment-free period for treatment-free males.
- The quantity of food consumed by each female was measured once a week, over a 7 day period, from the first day of treatment until the start of the mating period, during gestation for the intervals days 0-7, 7-14 and 14-20 post-coitum, and during lactation days 1-5 post-partum. Food consumption of treatment-free females was also measured during days 5-12 and 12-19 post-partum.
- Food consumption was not measured for males or females during the mating period.
- Food intake per animal and per day was calculated by noting the difference between the food given and that in the food hopper the next time.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: Day 5 post partum
- Organs examined: All animals were observed for macroscopic lesions
The kidneys, livers and spleens were weighed for all animals.
The mammary gland area, ovaries (with oviducts), uterus (horns and cervix), vagina, kidneys, liver and spleen were preserved for all animals.
Microscopic examinations of the ovaries, kidneys and livers were performed for all control and high dose animals. - Ovaries and uterine content:
- Special attention was paid to the reproductive organs. The numbers of corpora lutea and implantation sites were also recorded for females sacrificed as scheduled on Day 6 p.p. and on Day 20 p.p. (except the number of corpora lutea on this latter day) and for females sacrificed on Days 25-26 p.c. due to no delivery.
For non-pregnant females the absence of implantation scars on the uterus was checked using the ammonium sulphide staining technique (Salewski, 1964). - Statistics:
- Data were expressed as group mean values ± standard deviation (body weight, body weight change, food consumption, number of corpora lutea, number of implantation sites, number of pups and pup body weight , gestation length) or as ratios (pre-implantation loss, post-implantation loss, pup observations, mating index, fertility index, gestation index, live birth index, viability index).
Data for non-pregnant females are not included in group mean calculations such as body weight, body weight change, food consumption
Body weight, food consummation and reproductive data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (ratios).
Organ weight data was assed using PathData software (level of significance 0.05 or 0.01) according to the following:
Kolmogorov test for normality was performed on each group. Where evidence for non-normal distribution occurred in one group or more, non-parametric test for groups differences (Kruskal-Wallis) was performed if the number of groups was greater than 2. The data were then reviewed using Dunn’s test when evaluating more than 2 groups, or Wilcoxon test, where 2 groups were examined.
If the Kolmogorov test for normality did not demonstrate non-normal distribution occurred in one group or more, the data were analysed of homogeneity of variance (Bartlett test/ F-test). Where a high homogeneity of variance was observed, the data were evaluated using a parametric test for group differences in means using one-way analysis of variance if more than 2 groups. Further analysis was performed using Dunnett’s test for more than 2 groups, or t-test if 2 groups. - Indices:
- Pre-implantation loss: ((Number of corpora lutea – Number of implantation sites)/Number of corpora lutea) x 100
Post implantation loss (Calculated manually): ((Number of implantation sites – Number of live pups)/Number of implantation sites) x 100
Mating index: (Number of mated animals/Number of paired animals) x 100
Fertility index: (number of pregnant female partners/number of mated pairs) x 100
Gestation index: (Number of females with live born pups/Number of pregnant females) x 100
Live birth index: (Number of live born pups/Number of delivered pups) x 100
Viability index and Day 4 p.p.: (Number of surviving pups on Day 4 p.p./Number of live born pups) x 100
Results and discussion
Results: maternal animals
General toxicity (maternal animals)
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - The only test item-related clinical sign was ptyalism noted in 4/15 males at 1000 mg/kg bw/day. In view of the low incidence (4/30 animals in the high dose group) and the absence of a similar finding in females, it was considered to be of no toxicological significance.
- Other clinical signs recorded in test item-treated groups (ptyalism, chromodacryorrhea, hair loss, cutaneous lesion, reddish vaginal discharge, piloerection, pallor) were not attributed to the test item treatment as there was no dose relationship and observations were recorded in only a few animals or in a similar incidence to that in controls. - Mortality:
- no mortality observed
- Description (incidence):
- There were no premature deaths in test item-treated groups.
Control female D27529 was prematurely sacrificed on day 22 post-coitum due to the presence of a mass in the urogenital region that made a normal delivery impossible. Swollen urogenital area was noted from day 12 post-coitum and piloerection, round back, hypoactivity and absence of feces were recorded on day 22 post-coitum. Another mas, in the thoracic region, was noted in this female from day 9 post-coitum. The two large masses correlated with mammary gland adenocarcinoma. This finding is not uncommon in female rats of this strain and age.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- BODY WEIGHT - Mean body weights and mean body weight changes (g) are summarised in the tables attached. - There were no effects on mean body weights and mean body weight changes in males at any dose levels during treatment and treatment-free periods. The statistically significant higher mean body weights gain at 250 mg/kg bw/day over the first 15 days of treatment in males was considered to be incidental and not dose-related. - There were no toxicologically significant effects on mean body weight in females at any dose levels during treatment and treatment-free periods. - Statistically significant lower mean body weight gains were recorded at 500 and 1000 mg/kg bw/day in the second week of treatment, and statistically significant higher mean body weight gains were recorded at 1000 mg/kg bw/day in the first week of the treatment-free period. As the effects were transient and did not have significant impact on mean body weights, they were considered to be of no toxicologicical significance.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- There were no toxicologically significant effects on mean food consumption during treatment and treatment-free periods.
At 1000 mg/kg bw/day, the statistically higher mean food consumption in females during the first week of treatment and the last week of gestation was considered to be of no toxicological significance. The increases were small, transient and more variable during the first week of treatment than in controls. Moreover, there was no relevant impact on mean body weights.
At 500 mg/kg bw/day in males, the statistically significant lower mean food consumption during the second week of treatment was not ascribed to treatment with the test item as it was not dose related. - 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
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- At the end of the treatment period there were test item-related liver and kidney mean weight differences in males
The final mean body weights were considered to be unchanged by test item administration.
Moderately increased, slightly dose-related, absolute and relative-to-body mean kidney weights were noted in males at all dose levels (up to +34 % in absolute weights with P < 0.01 in males at 1000 mg/kg bw/day. These changes were considered to be related to test item administration since they correlated with the hepatocellular hypertrophy and the increased periportal vacuolation seen microscopically.
It was considered that the minimally higher liver relative-to-body weights in females at 250 and 500 mg/kg bw/day were probably unrelated to test item administration since those changes were poorly dose related and of very low magnitude. The slides of liver from females were not examined. The minimal epididymides and spleen absolute and relative-to-body mean weight changes in males at all dose-levels were not considered to be test item-related because they were of small magnitude, not dose-related and uncorrelated with microscopic findings. At the end of the treatment-free period there were no mean liver weight differences while minimally increased kidney weights were seen in males previously treated at 1000 mg/kg bw/day (+17 % in relative-to-body weights with p < 0.05), with microscopic correlates as mentioned at the end of the treatment period. This suggested total reversibility for the liver changes but not for the kidney changes for which reversibility was incomplete. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- There was one unscheduled death when one control female was sacrificed with two large subcutaneous white masses (3 x 4 and 2 x 3 cm) which correlated with mammary gland adenocarcinoma.
At the end of the treatment period there were test item-related irregulat colour and/or tan discolouration in the kidneys from 6/10 males treated with 100 mg/kg bw/day and from all males treated at 250, 500 or 1000 mg/kg bw/day. These changes were considered to be related to test item administration since they correlated with the increased weights and hyaline droplets in tubular epithelium, tubular dilation and dilated tubules with eosinophilic content seen microscopically in the kidneys of these males.
There were increased incidence of accentuated lobular pattern in the liver from males treated at 100, 250, 500 or 1000 mg/kg bw/day, together with tan discolouration. These findings correlated with the increased weights and the hepatocellular hypertrophy plus the increased periportal vacuolation seen microscopically in these animals.
The few other gross observations were considered to be consistent with spontaneous findings encountered in the rats of this strain and age because they were not dose-related, isolated and/or correlated with common background lesions.
At the end of the treatment-free period there were no test item-related macroscopic findings, suggesting a complete reversibility of these changes - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- . Microscopic examination The control female sacrificed on day 38 had a mammary gland adenocarcinoma. This finding is not uncommon in female rats of this strain and age. At the end of the treatment period a series of dose-related test item-related microscopic findings were seen in the kidneys and liver of males treated from 100 mg/kg bw/day, which were considered adverse at 1000 mg/kg bw/day.
Kidney - Minimal degeneration/necrosis in the kidneys from two males treated at 1000 mg/kg bw/day, suggesting an adverse effect at this dose level. - Increased incidence and severity of minimal to moderate tubular basophilia at all dose levels, considered not to be adverse in view of the low magnitude of the increase. - Minimal to marked hyaline droplets in tubular epithelium in all test item-treated males, considered to be a species-specific change not relevant in humans. - Minimal to slight tubular dilation (i.e. with empty lumen), together with minimal to moderate dilated tubules containing a granular eosinophilic content, considered not to be adverse in view of the absence of associated degeneration/necrosis. - Increased incidence of minimal infiltrate of mononuclear cells in males treated at 1000 mg/kg bw/day although poorly dose-related, considered not to be adverse in view of the minimal severity of this change.
Liver - Minimal hepatocellular hypertrophy, considered not to be adverse in view of the minimal severity of this change. - Increased incidence and severity of minimal to slight periportal vacuolation, considered not to be adverse in view of the low magnitude of this increase. These findings correlated with gross irregular colour and tan discolouration in kidneys and accentuated lobular pattern with tan discolouration in liver, together with increased weights. There were no test item-related findings in the testes, epididymides or ovaries at microscopic examination. There were a few other microscopic observations, which were seen at low incidence, at minimal severity, were not dose-related and correlated with background lesions seen in rats of this strain and age. At the end of the treatment-free period there were no test item-related findings in the liver while lesions similar to those recorded at the end of the treatment period were seen in the kidneys and correlated with the increased mean renal weights. - Increased incidence and severity of slight to marked tubular basophilia. - Minimal hyaline droplets in tubular epithelium from one test item-treated male. - Increased severity of minimal to moderate tubular dilation (i.e. with empty lumen) together with slight to moderate dilated tubules containing a granular eosinophilic content. - Increased incidence of minimal infiltrate of mononuclear cells. These findings suggested complete reversibility for the liver changes while there was incomplete reversal of the renal findings. - Histopathological findings: neoplastic:
- no effects observed
Maternal developmental toxicity
- Pre- and post-implantation loss:
- no effects observed
- Early or late resorptions:
- no effects observed
- Description (incidence and severity):
- - Control females D27525, D27526 and D27533 and female D27544 from the low dose group were sacrificed on days 25/26 post-coitum because they did not deliver (not ascribed to test item treatment for D27544 and the other animals were controls). The animals were found to be non-pregnant except control female D27525 which had one late resorption in the uterus at necropsy. There were no test item-related effects on mean delivery data at any dose levels.
- Changes in pregnancy duration:
- not specified
- Description (incidence and severity):
- Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed - Changes in number of pregnant:
- no effects observed
- Description (incidence and severity):
- There were no effects on mean pairing, mating and fertility data at any dose level.
- Details on maternal toxic effects:
- Maternal toxic effects:no effects
- The higher mean number of corpora lutea at 500 mg/kg bw/day was due to female D27557 with an excessive number of corpora lutea. As this was isolated, the finding was considered to be incidental. Nevertheless, the very high number of corpora lutea (40) likely did not correspond to gestational corpora lutea only (total number abnormally high).
-Female D27537 (100 mg/kg bw/day) and females D27557 and D27564 (500 mg/kg bw/day) were blocked in diestrous during most of the mating period and thus took more days to mate compared with the other animals. This was not considered to be related to test item treatment as it was not dose related and occurred in a limited number of animals.
Effect levels (maternal animals)
open allclose all
- Dose descriptor:
- NOAEL
- Remarks:
- females
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: other:
- Dose descriptor:
- NOEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
Maternal abnormalities
- Key result
- Abnormalities:
- no effects observed
Results (fetuses)
- Fetal body weight changes:
- no effects observed
- Description (incidence and severity):
- There were no toxicologically significant effects on pup mean body weight and mean body weight gains at any dose-levels during treatment and treatment-free periods.
At 1000 mg/kg/day and when compared with controls, the slightly higher mean body weight gains and mean body weights during the treatment-free period was mainly due to the lower mean number of pups kept after the end of treatment of their mothers. - Reduction in number of live offspring:
- no effects observed
- Changes in sex ratio:
- no effects observed
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:no effects
Effect levels (fetuses)
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: absence of adverse effects in pups at all dose levels
- Remarks on result:
- not determinable due to absence of adverse toxic effects
Fetal abnormalities
- Abnormalities:
- not specified
Overall developmental toxicity
- Key result
- Developmental effects observed:
- no
Any other information on results incl. tables
CHEMICAL
ANALYSIS OF DOSE FORMULATIONS
-
Test item concentrations in the administered dose formulations analysed
in weeks 1, 3 and 6 were within an acceptable range of variation (-4.9 %
to +3.9%) when compared to nominal values (± 10 % of nominal
concentration).
-
No test item was observed in the control dose formulation in week 1
although a small peak (below the limit of quantification) was observed
in weeks 3 and 6.
Table 1: Parental Bodyweight and Mean Body Weight Changes (g)
Sex |
Male |
Female |
||||||||
Dose level (mg/kg bw/day) |
0 |
100 |
250 |
500 |
1000 |
0 |
100 |
250 |
500 |
1000 |
Premating or whole study |
428 |
430 |
416 |
409* |
423 |
252 |
255 |
251 |
255 |
259 |
Day 1 |
462 |
470 |
458 |
450 |
463 |
263 |
266 |
259 |
265 |
273 |
Day 8 |
495 |
506 |
493 |
484 |
494 |
282 |
279 |
273 |
275 |
282 |
Day 15 |
506 |
520 |
509 |
497 |
510 |
/ |
/ |
/ |
/ |
/ |
Day 22 |
526 |
546 |
538 |
525 |
532 |
/ |
/ |
/ |
/ |
/ |
Day 29 |
535 |
546 |
535 |
522 |
534 |
/ |
/ |
/ |
/ |
/ |
Day 35 |
580 |
/ |
/ |
/ |
553 |
/ |
/ |
/ |
/ |
/ |
Day 43 (treatment free) |
566 |
/ |
/ |
/ |
545 |
/ |
/ |
/ |
/ |
/ |
Day 50 (treatment free) |
+34 |
+40 |
+42 |
+41 |
+40 |
+10 |
+11 |
+8 |
+10 |
+14 |
Days 1-8 |
+33 |
+36 |
+35 |
+34 |
+31 |
+19 |
+13 |
+14 |
+10* |
+9** |
Days 8-15 |
+67 |
+76 |
+77* |
+75 |
+71 |
+29 |
+23 |
+22 |
+20 |
+23 |
Days 15-22 |
+11 |
+14 |
+16 |
+13 |
+16 |
/ |
/ |
/ |
/ |
/ |
Days 22-29 |
+20 |
+26 |
+29 |
+28 |
+23 |
/ |
/ |
/ |
/ |
/ |
Days 29-36 |
+9 |
0 |
-3 |
-3 |
+2 |
/ |
/ |
/ |
/ |
/ |
Day 15-36 |
+40 |
+40 |
+42 |
+38 |
+41 |
/ |
/ |
/ |
/ |
/ |
Days 1-36 |
+107 |
+116 |
+119 |
+114 |
+111 |
/ |
/ |
/ |
/ |
/ |
Day 36-43 |
+13 |
/ |
/ |
/ |
+13 |
/ |
/ |
/ |
/ |
/ |
Days 43-50 |
-13 |
/ |
/ |
/ |
-8 |
/ |
/ |
/ |
/ |
/ |
Days 36-50 |
-1 |
/ |
/ |
/ |
+4 |
/ |
/ |
/ |
/ |
/ |
Days 1-50 |
+134 |
/ |
/ |
/ |
+131 |
/ |
/ |
/ |
/ |
/ |
Table 2:Female bodyweight changes Day 0 p.c. to Day 19 p.p.
Dose level (mg/kg bw/day) |
0 |
100 |
250 |
500 |
1000 |
Gestation |
|||||
Day 0 p.c. |
284 |
287 |
276 |
286 |
282 |
Day 7 p.c. |
318 |
323 |
314 |
318 |
321 |
Day 14 p.c. |
358 |
366 |
357 |
361 |
363 |
Day 20 p.c. |
437 |
452 |
452 |
452 |
449 |
Day 0-7 p.c. |
+34 |
+35 |
+35 |
+31 |
+38 |
Day 7-14 p.c. |
+39 |
+43 |
+43 |
+43 |
+43 |
Day 14-20 p.c. |
+80 |
+86 |
+86 |
+91 |
+86 |
Day 0-20 p.c. |
+153 |
+164 |
+164 |
+165 |
+167 |
Lactation |
|||||
Day 1 p.p |
351 |
355 |
341 |
345 |
346 |
Day 5 p.p |
358 |
365 |
356 |
351 |
349 |
Day 12 p.p (treatment free) |
377 |
/ |
/ |
/ |
380 |
Day 19 p.p (treatment free) |
383 |
/ |
/ |
/ |
371 |
Days 1-5 p.p |
+7 |
+10 |
+15 |
+6 |
+3 |
Days 5-12 p.p |
+29 |
/ |
/ |
/ |
+49* |
Days 12-19 p.p |
+5 |
/ |
/ |
/ |
-9 |
Days 5-19 p.p |
+34 |
/ |
/ |
/ |
+41 |
Days 1-19 p.p. |
+35 |
/ |
/ |
/ |
+39 |
* p < 0.05 |
Table 3: Meanparental food consumption (g)
Sex |
Male |
Female |
||||||||
Dose level (mg/kg bw/day) |
0 |
100 |
250 |
500 |
1000 |
0 |
100 |
250 |
500 |
1000 |
Pre-mating or whole study |
||||||||||
Days 1-8 |
31 |
32 |
32 |
31 |
33 |
19 |
20 |
19 |
20 |
23* |
Days 8-15 |
31 |
30 |
30 |
29** |
30 |
21 |
20 |
19 |
19 |
20 |
Days 36-43 (treatment free) |
33 |
/ |
/ |
/ |
31 |
/ |
/ |
/ |
/ |
/ |
Days 43-50 (treatment free) |
29 |
/ |
/ |
/ |
28 |
/ |
/ |
/ |
/ |
/ |
Gestation |
||||||||||
Days 0-7 |
/ |
/ |
/ |
/ |
/ |
23 |
23 |
21 |
22 |
24 |
Days 7-14 |
/ |
/ |
/ |
/ |
/ |
25 |
25 |
24 |
25 |
25 |
Days 14-20 |
/ |
/ |
/ |
/ |
/ |
29 |
29 |
29 |
30 |
32* |
Lactation |
||||||||||
Days 1-5 |
/ |
/ |
/ |
/ |
/ |
39 |
40 |
39 |
39 |
36 |
Days 5-12 (treatment free) |
/ |
/ |
/ |
/ |
/ |
77 |
/ |
/ |
/ |
71 |
Days 12-19 (treatment free) |
/ |
/ |
/ |
/ |
/ |
99 |
/ |
/ |
/ |
87 |
* p < 0.05, ** p < 0.01 |
Table 4: Mean organ weight changes (g)
Sex |
Male |
Female |
||||||
Dose level (mg/kg bw/day) |
100 |
250 |
500 |
1000 |
100 |
250 |
500 |
1000 |
Examined/Number of animals |
10/10 |
10/10 |
10/10 |
10/10 |
10/10 |
10/10 |
10/10 |
10/10 |
Final bodyweight |
+5 |
+3 |
+1 |
+2 |
-2 |
-5 |
-4 |
-3 |
-Liver |
||||||||
Absolute |
+10* |
+10* |
+7 |
+13** |
+1 |
+4 |
+6 |
+6 |
Relative |
+5 |
+7* |
+7* |
+11** |
+3 |
+10* |
+11** |
+9 |
-Kidneys |
||||||||
Absolute |
+23** |
+24** |
+22** |
+34** |
+6 |
+5 |
+6 |
+2 |
Relative |
+18* |
+20** |
+22** |
+31** |
+7 |
+10* |
+10** |
+5 |
* p < 0.05, ** p < 0.01 |
Table 5: Mean organ weight changes (g) treatment-free
Sex |
Male |
Female |
Dose level (mg/kg bw/day) |
1000 |
1000 |
Examined/Number of animals |
5/5 |
5/5 |
Final bodyweight |
-4 |
-2 |
-Kidneys |
||
Absolute |
+12 |
-1 |
Relative |
+17* |
+1 |
* p < 0.05 |
Table 6: Parental animals - gross findings at necropsy
Sex |
Males |
||||
Dose-level (mg/kg bw/day) |
0 |
100 |
250 |
500 |
1000 |
Number of animals |
10 |
10 |
10 |
10 |
10 |
Kidney, irregular colour |
0 |
0 |
1 |
6 |
3 |
kindey tan discolouration |
0 |
6 |
9 |
4 |
7 |
liver, accentuated lobular pattern |
3 |
6 |
7 |
7 |
9 |
liver, tan discolouration |
0 |
2 |
1 |
0 |
1 |
Table 7: Parental microscopic findings
Sex |
Male |
||||
Dose-level (mg/kg bw/day) |
0 |
100 |
250 |
500 |
1000 |
Number of animals |
10 |
10 |
10 |
10 |
10 |
Kidney; degeneration/necrosis; tubule (grade 1 minimal) |
0 |
0 |
0 |
0 |
2 |
Kidney; basophilia; tubule (grade 1 minimal) |
5 |
2 |
0 |
0 |
0 |
Kidney; basophilia; tubule (grade 2 slight) |
1 |
4 |
1 |
1 |
1 |
Kidney; basophilia; tubule (grade 3 moderate) |
0 |
4 |
9 |
9 |
9 |
Kidney; dilation; tubule (grade 1 minimal) |
0 |
5 |
7 |
9 |
4 |
Kidney; dilation; tubule (grade 2 slight) |
0 |
3 |
0 |
1 |
6 |
Kidney; dilated tubules with eosinophilic content (grade 1 minimal) |
0 |
0 |
4 |
1 |
0 |
Kidney; dilated tubules with eosinophilic content (grade 2 slight) |
0 |
2 |
4 |
4 |
9 |
Kidney; dilated tubules with eosinophilic content (grade 3 moderate) |
0 |
2 |
1 |
4 |
1 |
Kidney; hyaline droplets; tubular epithelium (grade 1 minimal) |
0 |
1 |
0 |
0 |
0 |
Kidney; hyaline droplets; tubular epithelium (grade 2 slight) |
0 |
3 |
0 |
1 |
0 |
Kidney; hyaline droplets; tubular epithelium (grade 3 moderate) |
0 |
5 |
9 |
3 |
5 |
Kidney; hyaline droplets; tubular epithelium (grade 4 marked) |
0 |
1 |
1 |
6 |
5 |
Kidney; Infiltrate; mononuclear cell infiltrate (grade 1 minimal) |
1 |
5 |
2 |
1 |
7 |
Liver; hypertrophy; hepatocellular (grade 1 minimal) |
0 |
1 |
4 |
5 |
6 |
Liver; vacuolation; periportal (grade 1 minimal) |
2 |
7 |
6 |
4 |
7 |
Liver; vacuolation; periportal (grade 2 slight) |
0 |
0 |
1 |
3 |
1 |
Table 8: Parental microscopic findings (treatment-free period)
Sex |
Male |
|
Dose-level (mg/kg bw/day) |
0 |
1000 |
Number of animals |
10 |
10 |
Kidney; basophilia; tubule (grade 1 minimal) |
3 |
0 |
Kidney; basophilia; tubule (grade 2 slight) |
0 |
2 |
Kidney; basophilia; tubule (grade 3 moderate) |
0 |
2 |
Kidney; basophilia; tubule (grade 4 marked) |
0 |
1 |
Kidney; dilation; tubule (grade 1 minimal) |
1 |
1 |
Kidney; dilation; tubule (grade 2 slight) |
0 |
1 |
Kidney; dilated tubules with eosinophilic content (grade 2 slight) |
0 |
3 |
Kidney; dilated tubules with eosinophilic content (grade 3 moderate) |
0 |
2 |
Kidney; hyaline droplets; tubular epithelium (grade 1 minimal) |
0 |
1 |
Kidney; Infiltrate; mononuclear cell infiltrate (grade 1 minimal) |
1 |
3 |
Table 9: Reproductive and delivery data
Dose level (mg/kg bw/day) |
0 |
100 |
250 |
500 |
1000 |
Number of animals paired (M+F) |
15+15 |
10+10 |
10+10 |
10+10 |
15+15 |
Number of males mated |
15 |
10 |
10 |
10 |
15 |
Number of females mated |
15 |
10 |
10 |
10 |
15 |
Mating index (%) |
100 |
100 |
100 |
100 |
100 |
Mean number of days taken to mate |
2.2 |
3.5 |
2.2 |
4.2 |
2.5 |
Number of pregnant females |
13 |
9 |
10 |
10 |
15 |
Fertility index (%) |
86.7 |
90 |
100 |
100 |
100 |
Number of females with live born pups |
11 |
9 |
10 |
10 |
15 |
Gestation index (%) |
84.6 |
100 |
100 |
100 |
100 |
Number of females which delivered |
11 |
9 |
10 |
10 |
15 |
Mean duration of gestation (days) |
22 |
22 |
21.9 |
22 |
21.9 |
Mean number of corpora lutea |
15 |
15.6 |
15.6 |
18.3 |
16.5 |
Mean number of implantations |
15 |
15.6 |
15.6 |
16 |
15.5 |
Mean pre-implantation loss (%) |
13.3 |
0 |
0 |
5.8 |
0.8 |
Mean number of pups delivered |
12.2 |
12.8 |
14.5 |
13.4 |
13.3 |
Mean post-implantation loss (%) |
17.6 |
18 |
6.4 |
15.6 |
13.3 |
Table 10: Pup viability indices and sex ratio
Dose level (mg/kg bw/day) |
0 |
100 |
250 |
500 |
1000 |
Viability index on Day 4 p.p. (%) |
97.8 |
95.7 |
91 |
96.3 |
93 |
No. of pups cannibalized |
0 |
1 |
3 |
1 |
10 |
No. of pups found dead |
4 |
4 |
10 |
4 |
4 |
Sex ratio at birth (% males) |
46.3 |
53.5 |
62.8 |
47 |
50 |
Table 11:Pups Bodyweight (g)
Sex |
Male |
Female |
||||||||
Dose level (mg/kg bw/day) |
0 |
100 |
250 |
500 |
1000 |
0 |
100 |
250 |
500 |
1000 |
Body weight |
||||||||||
Day 1 p.p. |
8.2 |
7.8 |
7.3 |
7.7 |
7.7 |
7.7 |
7.3 |
6.9 |
7.3 |
7.4 |
Day 5 p.p. |
14.1 |
13.8 |
12.7 |
12.9 |
13 |
13.6 |
13.3 |
12.1 |
12.2 |
12.5 |
Day 12 p.p. (treatment free) |
26.2 |
/ |
/ |
/ |
29.2 |
25.6 |
/ |
/ |
/ |
26.2 |
Day 19 p.p. (treatment free) |
42.8 |
/ |
/ |
/ |
48.7 |
41.2 |
/ |
/ |
/ |
46.5 |
Body weight change |
||||||||||
Days 1-5 p.p. |
6 |
6 |
5.4 |
5.2 |
5.3 |
5.9 |
5.9 |
5.2 |
5 |
5.2 |
Days 5-19 p.p. (treatment free) |
30 |
/ |
/ |
/ |
35.6 |
29 |
/ |
/ |
/ |
33.9 |
Days 1-19 p.p. |
35.2 |
/ |
/ |
/ |
41.3 |
33.9 |
/ |
/ |
/ |
39.4 |
Table 12:Pup clinical observations
Dose level (mg/kg bw/day) |
0 |
100 |
250 |
500 |
1000 |
Haematoma on head |
1 (1) |
2 (1) |
|||
scab on back |
1 (1) |
||||
wound on urogenital area |
2 (1) |
||||
Dehydration |
1 (1) |
||||
Thin appearance |
1 (1) |
||||
Difficult breathing |
1 (1) |
||||
Cold to the touch |
1 (1) |
Table 13:Pup macroscopic observations
Dose (mg/kg bw/day) |
0 |
100 |
250 |
500 |
1000 |
Number of pup (litter) examined |
130 (11) |
110 (9) |
132 (10) |
129 (10) |
186 (15) |
Pale liver |
1 (1) |
1 (1) |
|||
Dilated renal pelvis |
1 (1) |
||||
Absent kidney and ureter |
1 (1) (treatment-free period) |
Applicant's summary and conclusion
- Conclusions:
- The No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 500 mg/kg bw/day for males based on the adverse microscopic effects in kidneys seen at the higher dose and 1000 mg/kg bw/day for females based on the absence of adverse effects in females. However, the renal effects seen in male rats are not relevant to human exposure. The No Observed Effect Level (NOEL) for parental reproductive performance was considered to be 1000 mg/kg bw/day based on the absence of effects in mean reproductive data at all dose levels. The No Observed Adverse Effect Level (NOAEL) for toxic effects on progency was considered to be 1000 mg/kg bw/day based on absence of adverse effects in pups at all dose levels.
- Executive summary:
GUIDELINE
The study design was based on OECD 421 of 27 July 1995. The objective was to evaluate the portential toxic effects of the test item following daily oral (gavage) administration to male and female rats from before mating, through mating and, for females, through gestation until day 5 post-partum. On completion of the treatment period, additional dams and litters were held for a two week treatment-free period in the control and high dose groups in order to evaluate the reversibility of any findings. The study provided initial information on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of the conceptus and partrutition, and on reversibility of potential findings on development of the offspring or gonadal weight and morphology.
METHODS
Four groups of 10 male and 1o female (groups 2 to 4) or 15 male and 15 female (group 5) Sprague-Dawley rats received the test item daily by oral (gavage) administration before mating, through mating and, for females, through gestation until day 5 post-partum. The test item was administered as a solution in the vehicle, corn oil, at dose levels of 100, 250, 500 and 1000 mg/kg bw/day. Another group of 15 males and 15 females received the vehicle alone, under the same experimental conditions, and acted as the control group. A constant dose volume of 4 mL/kg bw/day was used. On completion of the treatment period, the animals in each group were sacrificed, except for five animals per sex in the control and high dose groups which were kept for a 2 -week recovery period. The concentrations of the dose formulations were checked in study weeks 1, 3 and 6.
The animals were checked twice daily for mortality and morbidity and once daily for clinical signs during the treatment and treatment-free periods. Body weight and food consumption were recorded once a week during pre-mating and mating periods (food consumption not during mating), and during gestation on days 0, 7, 14 and 20 post-coitum plus lactation on days 1 and 5 post-partum. These data were also recorded on days 12 and 19 post-coitum for treatment-free females and weekly after the mating period for treatment-free males.
Animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until day 5 post-partum or until day 19 post-partum for treatment-free females. The total litter sizes and number of pups of each sex were recorded after birth. Pups were observed daily for clinical signs, abnormal behaviour and external abnormalities and weighed on days 1 and 5 post-partum. Pups of treatment-free females were also weighed on days 12 and 19 post-partum.
Males were sacrificed after at least 5 weeks of treatment and females on day 6 post-partum except for treatment-free animals, which were sacrificed 2 weeks after the end of treatment (thus on day 20 post-partum for females). Final body weights and selected organ weights (epididymides, testes, kidneys, liver, spleen) were recorded and a macroscopic post-mortem examination of the prinicpal thoracic and abdominal organs was performed. Particular attention was paid to the reproductive organs. A microscopic examination was performed on ovaries(with oviducts) from all females sacrificed on day 6 post-partum in the control and high dose groups and epididymides and/or testes from all males sacrificed at the end of the treatment period in the control and high dose groups. A microscopic examination was also performed on the kidneys and liver from all males sacrificed at the end of the treatment or treatment-free period in all groups and on all macroscopic lesions. Pups were sacrificed on day 5 post-partum (or day 19 post-coitum for treatment-free pups) and submitted for post-mortem examination of the principal thoracic and abdominal organs.
RESULTS
Chemical analysis: No test item was detectable above the limit of quantification in the control dose formulation and the test item concentrations in the analysed dose formulations were within an acceptable range.
F0 animals: The following findings were reported:
- No premature deaths in the test-item treated groups during treatment and treatment-free periods.
- No toxicologically significant clinical signs during treatment and treatment-free periods.
- No toxicologically significant effects on mean body weights, mean body weight changes and mean food consumption during treatment and treatment-free periods.
- No effects on mean pairing, mating, fertility and delivery data.
- Test item-related kidney and liver mean weight increases were seen in males treated with 100 mg/kg bw/day or above.
- Test item-related irregular colour and or tan discolouration of kidneys and increased incidence of accentuated lobular pattern together with tan discolouration was observed in males treated with 100 mg/kg bw/day or above. At the end of the treatment-free period, there were no test item-related gross findings in either the liver or the kidneys.
- A series of test-item related microscopic findings was found in the kidneys and liver of males treated with 100 mg/kg bw/day or above. These findings were degradation/necrosis of tubules (adverse) in 2/10 males treated at 1000 mg/kg bw/day, hyaline droplets in tubular epithelium, tubular dilation with or without eosinophilic content in kidneys, and hepatocellular hypertrophy plus increased periportal vacuolation in the liver. At the end of the treatment-free period, the reversibility of the liver changes was complete whilst reversibility of kidney changes was incomplete.
Pups: The following findings were reported:
- No test item-related effects on pup viability during treatment and treatment-free periods.
- No test-item related clinical signs during treatment and treatment-free periods.
- No toxicologically significant effects on mean body weight and mean body weight gains during treatment and treatment-free periods.
- No clear indication of a test item treatment effect on percentage of male pups at birth.
- No test item-related necropsy findings during treatment and treatment-free periods.
CONCLUSION
The No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 500 mg/kg bw/day for males based on the adverse microscopic effects in kidneys seen at the higher dose and 1000 mg/kg bw/day for females based on the absence of adverse effects in females. However, the renal effects seen in male rats are not relevant to human exposure. The No Observed Effect Level (NOEL) for parental reproductive performance was considered to be 1000 mg/kg bw/day based on the absence of effects in mean reproductive data at all dose levels. The No Observed Adverse Effect Level (NOAEL) for toxic effects on progency was considered to be 1000 mg/kg bw/day based on absence of adverse effects in pups at all dose levels.
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