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EC number: 266-120-2 | CAS number: 66072-08-0
- 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
![](https://echa.europa.eu/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 23 July 2012 to 2 October 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EPA OPPTS 870.3650
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Strain: Crl:WI(Han) (outbred, SPF-Quality).
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: The average weight of the males was 326 g and the average weight of the females was 206 g on Day 1 of the study.
- Housing: Pre-mating, animals were housed in groups of 5 animals/sex/cage in plastic cages (height 18 cm). During mating, females were caged together with males on a one-to-one-basis in plastic cages (height 18 cm). Post-mating, males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in plastic cages (height 18 cm). During lactation, pups were kept with the dam until termination in plastic cages (height 18 cm).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet.
- Water (e.g. ad libitum): Water Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24 °C
- Humidity (%): 40 to 70 % relative
- Air changes (per hr): approximately 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark cycle
IN-LIFE DATES: From: 9 August 2012 To: 2 October 2012 - Route of administration:
- oral: gavage
- Vehicle:
- propylene glycol
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS
VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
- Specific gravity: 1.036
- Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenised to a visually acceptable level. No correction was made for the purity of the test material. Adjustment was made for specific gravity of the vehicle. Formulations were placed on a magnetic stirrer during dosing. - Details on mating procedure:
- Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The purpose of the analytical phase was to determine the accuracy of preparation, homogeneity and stability of the test material in formulations.
Samples of dose preparations were taken on a single occasion during the treatment phase and were stored and dispatched on dry ice to the test site for formulation analysis.
Formulations were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in the vehicle over 5 hours was also determined (highest and lowest concentration).
Specific sampling instructions:
− Appearance of the formulations was recorded prior to sampling
− Formulations were placed on a magnetic stirrer during sampling
− Sampling was conducted with a clean pipette tip used for every group
− Duplicate samples were taken from each sampling height, with a sample volume of 0.5 mL
− Samples were weighed (grams) on an analytical balance at a precision of 4 decimals
− T, M and B samples were stored on dry ice immediately after sampling
− S (Stability) samples were kept at room temperature under normal laboratory light conditions for
5 hours, and then placed on dry ice
− All samples were transferred to the weighing room at once for dispatch
SAMPLES
In total, 20 samples were included, randomised over 4 formulation groups. Group 1 was the control group. Group 2, 3 and 4 were dosed at 75, 250 and 750 mg/kg bw/day, respectively. The samples of the control group and group 3 were taken in duplicate from the middle position of the container and immediately stored on dry ice.
Samples of treatment groups 2 and 4 were taken in duplicate from the top, middle and bottom position of the container. In addition, stability samples were taken from formulation groups 2 and 4, in duplicate at the middle position of the container, stored for 5 hours at room temperature under normal laboratory light conditions and then placed on dry ice.
The samples were stored at the analytical facility at a target temperature ≤-70 °C.
ANALYTICAL METHOD
The determination was performed using LCMS/MS.
SAMPLE INJECTIONS
The analytical run was acquired in the following order:
- Analytical blank sample
- Calibration curve
- Analytical blank sample
- Analytical blank sample
- Procedural recovery sample low and high (replicate 1)
- Analytical samples
- Stability at storage conditions low and high (replicate 1 and 2)
- Procedural recovery samples low and high (replicate 2)
Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were
analysed by single injection.
ELECTRONIC DATA CAPTURE
System control, data acquisition and data processing were performed using the program Analyst version 1.5.2 (Applied Biosystems, Burlington, Canada).
Electronic raw data was stored in the document management system using KnowledgeTree version 3.5.2c (Raleigh, USA).
FORMULAS
Response (Y)
Peak area test substance [cps]
Calibration curve
Y = bX + a
where:
X = nominal concentration [mg/L]
b = slope [cps × L/mg]
a = intercept [cps]
Analysed concentration (X)
X = ((Y - a) / b) x ((V x d) / w [mg/g]
where:
w = weight sample [mg]
V = volume volumetric flask [mL]
d = dilution factor
(Y - a) / b values are obtained from Analyst
Recovery
(Analysed concentration [mg/g] / Nominal concentration [mg/g]) x 100 [%]
Accuracy
(Analysed concentration [mg/g] / Target concentration [mg/g]) x 100 [%]
Relative difference (relative diff.)
((Ct - C0) / C0) x 100 [%]
where:
Ct = mean concentration of stored samples [mg/g]
C0 = mean concentration of non-stored samples [mg/g]
SPECIFICATIONS
Run Acceptance
Calibration curves with a coefficient of correlation (r) of >0.99 and accuracies of the back calculated values of the calibration standard solutions in the range 85 to 115 % were accepted.
The mean recovery of the procedural recovery samples at each level had to be in the range 90 to 110 %.
The response in all analytical blank samples at the retention time of the test substance should be less than or equal to 30 % compared to the response of the lowest calibration standard (mean response of the duplicate injections).
Acceptance criteria formulations
Preparation of formulations was considered acceptable if the mean accuracy was in the range 90 to 110 % of the target concentration and if the coefficient of variation was ≤ 10 %. Formulations were considered stable if the absolute relative difference between the stored and freshly taken samples was ≤ 10 %.
RESULTS
Calibration curves
Calibration curves were constructed using six concentrations. For each concentration, two responses were acquired. Linear regression analysis was performed using the least squares linear regression with a 1/concentration² weighting factor.
The coefficient of correlation (r) of the calibration curve was 0.9998.
Additionally, there was no response in all analytical blank samples at the retention time of the test material.
Procedural recovery samples
The mean recoveries of the procedural recovery samples fell within the criterion of 90 to 110 %. It demonstrated that the analytical method was adequate for the determination of the test material in the test samples.
Test samples
In the Group 1 formulation, no test material was detected. The concentrations analysed in the formulations of Group 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 90 and 110 %).
The formulations of Group 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10 %). Analysis of Group 2 and Group 4 formulations after storage yielded an absolute relative difference ≤ 10 %. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 5 hours.
Stability at storage conditions
It was observed that the mean accuracy was 94.9 and 98.2 % at the levels high and low, respectively, which is within the criteria set (mean accuracy is in the range 90 to 110 %). It was therefore concluded that the test material was stable in propylene glycol over a storage period of 29 days at a temperature of ≤-70 °C. - Duration of treatment / exposure:
- Males were exposed for 28 days: 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42 to 55 days: 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
- Frequency of treatment:
- Once daily
- Remarks:
- Doses / Concentrations:
0, 75, 250 and 750 mg/kg bw/day
Basis:
actual ingested - No. of animals per sex per dose:
- 10 animals per sex per dose
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Based on the results of a 10-day dose range finding study conducted at the testing laboratory.
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Parental animals were observed for mortality / viability at least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, detailed clinical observations were conducted for all animals after dosing (at no specific time point, but around the same time point for all animals). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded.
BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
FOOD CONSUMPTION: Yes
- Time schedule: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.
FOOD EFFICIENCY: No
WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
OTHER: Observations were carried out for haematology, clotting potential, clinical chemistry and neurobehavioural examinations. - Oestrous cyclicity (parental animals):
- The numbers of former implantation sites and corpora lutea were recorded for all paired females.
- Sperm parameters (parental animals):
- Parameters examined in male parental generation: testis and epididymis weight. From 5 selected males from the control and high dose group and all males suspected to be infertile, slides of the testes were prepared to examine staging of spermatogenesis.
- Litter observations:
- Each litter was examined to determine the following, if practically possible:
- Mortality/viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were conducted for all animals.
- Bodyweights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation. - Postmortem examinations (parental animals):
- SACRIFICE
All males and 5 randomly selected females/group were deprived of food overnight (maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised using isoflurane and subsequently exsanguinated.
GROSS NECROPSY
Necropsy of parental animals: All males and 5 selected females/group for full examination, though all animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. The numbers of former implantation sites and corpora lutea were recorded for all paired females.
HISTOPATHOLOGY / ORGAN WEIGHTS
Samples of the following tissues and organs were collected and fixed: adrenal glands, aorta, brain (cerebellum, mid-brain, cortex), caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, epididymides, eyes (with optic nerve (if detectable) and harderian gland), female mammary gland area, femur including joint, heart, ileum, jejunum, kidneys, lacrimal gland (exorbital), larynx, liver, lung, infused with formalin, lymph nodes (mandibular, mesenteric), nasopharynx, oesophagus, ovaries, pancreas, peyer's patches [jejunum, ileum] if detectable, pituitary gland, preputial gland, prostate gland, rectum, salivary glands (mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, midthoracic, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid if detectable, tongue, trachea, urinary bladder, uterus, vagina and all gross lesions.
Samples of the following tissues and organs were collected and fixed from all remaining animals and females which failed to deliver: cervix, clitoral gland, coagulation gland, epididymides, mammary gland area, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, vagina and all gross lesions.
Organ weights: The following organ weights and terminal body weight were recorded from the 5 randomly selected animals/sex/group on the scheduled day of necropsy: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus (including cervix), prostate, seminal vesicles including coagulating glands and thyroid including parathyroid.
The following organ weights and terminal body weight were recorded from all remaining males: epididymides and testes.
All organ and tissue samples were processed, embedded and cut at a thickness of 2 to 4 micrometers and stained with haematoxylin and eosin. From the selected 5 males of the control and high dose group and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3 to 4 micrometers, and stained with PAS/haematoxylin.
A peer review on the histopathology data was performed by a second pathologist.
The following slides were examined by a pathologist:
- The preserved organs and tissues of the randomly selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis.
- The preserved organs and tissues of the animals of all dose groups which died spontaneously or were killed in extremis.
- All gross lesions of all animals (all dose groups).
- Liver (both sexes), kidney (males) and thyroids (females) of the 5 randomly selected animals of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
- The reproductive organs* of all males that failed to sire and all females that failed to deliver healthy pups. Additionally, the reproductive organs from two males were examined since the female with whom they were paired were killed in extremis or died spontaneously.
* Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina. - Postmortem examinations (offspring):
- SACRIFICE
Pups surviving to planned termination were killed by decapitation on Days 5 to 7 of lactation.
GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. - Statistics:
- The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to- one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
The following additional methods of statistical analysis were used:
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. - Reproductive indices:
- For each group, the following calculations were performed:
- Mating index (%) = (Number of females mated / Number of females paired) x 100
- Fertility index (%) = (Number of pregnant females / Number of females paired) x 100
- Conception index (%) = (Number of pregnant females / Number of females mated) x 100
- Gestation index (%) = (Number of females bearing live pups / Number of pregnant females) x 100
- Duration of gestation = Number of days between confirmation of mating and the beginning of parturition - Offspring viability indices:
- For each group, the following calculations were performed:
- Percentage live males at first litter check = (Number of live male pups at first litter check / Number of live pups at first litter check) x 100
- Percentage live females at first litter check = (Number of live female pups at first litter check / Number of live pups at first litter check) x 100
- Percentage of postnatal loss Days 0-4 of lactation = (Number of dead pups on Day 4 of lactation / Number of live pups at first litter check) x 100
- Viability index = (Number of live pups on Day 4 post partum / Number of pups born alive) x 100 - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- see below
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- see below
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- see below
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- see below
- Other effects:
- not examined
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- Dose descriptor:
- NOAEL
- Effect level:
- 75 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: see 'Remark'
- Dose descriptor:
- NOAEL
- Effect level:
- 750 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No toxicologically relevant effects on reproductive parameters were noted.
- Remarks on result:
- other: Generation: Reproductive Parameters (migrated information)
- Clinical signs:
- no effects observed
- Mortality / viability:
- mortality observed, treatment-related
- Description (incidence and severity):
- see below
- Body weight and weight changes:
- no effects observed
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- not examined
- Reproductive effects observed:
- not specified
- Conclusions:
- Under the conditions of this study, no reproductive toxicity was observed up to the highest dose level tested. The NOAEL for reproductive parameters is therefore considered to be 750 mg/kg/day.
- Executive summary:
A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guidelines OECD 422 and EPA OPPTS 870.3650 under GLP conditions.
Four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test material at 0, 75, 250 and 750 mg/kg/day in propylene glycol. Males were exposed for 28 days (2 weeks prior to mating, during mating, and up to termination) and females were exposed for 41 to 55 days (2 weeks prior to mating, during mating, during postcoitum and during at least 4 days of lactation).
Animals were evaluated for mortality/viability, clinical signs, functional observations and locomotor activity, body weight and food consumption, clinical pathology, macroscopy at termination, organ weights and histopathology on a selection of tissues and reproduction/developmental parameters.
Under the conditions of this study, the NOAEL for parental repeated dose toxicity was determined to be 75 mg/kg bw/day.
No toxicologically relevant effects on reproductive parameters were noted. The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.
Under the conditions of this study, no reproductive toxicity was observed up to the highest dose level tested. The NOAEL for reproductive parameters is therefore considered to be 750 mg/kg/day.
Reference
One female at 250 mg/kg was euthanised in extremis after 26 days of treatment; no definitive cause of death could be determined.
One female at 750 mg/kg was found dead after 33 days of treatment. Macroscopic examination confirmed that her death was secondary to a gavage error, and was in no way related to treatment.
CLINICAL SIGNS
Clinical signs noted for the female at 250 mg/kg that was euthanised in extremis included lethargy, hunched posture, laboured or shallow respiration, rales, piloerection, lean appearance and salivation. These were noted for several days prior to her death.
There were no other toxicology relevant observations noted up to 750 mg/kg.
Salivation was noted for all males and females at 250 and 750 mg/kg. Salivation was also noted for all males and 3/10 females at 75 mg/kg. Salivation was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to the taste of the test material.
Pale faeces were transiently noted for 4 females at 750 mg/kg. No toxicological relevance was attributed to this finding as it was transient and was not accompanied by any other findings.
Incidental findings that were noted included scabs and swelling of the right ear noted for control animals.
FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals. The variation in motor activity did not indicate a relation with treatment.
All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.
BODYWEIGHTS
Males at 750 mg/kg had significantly lower body weight gains on Days 8 and 14 of the mating period. As the difference from controls was only slight, this was not considered to be adverse.
All other body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
FOOD CONSUMPTION
There were no toxicologically relevant effects on absolute or relative food consumption noted up to750 mg/kg.
There was statistically significant increase in absolute food consumption for females at 750 mg/kg over post coitum Days 4 to 7. Absolute and relative food consumption was significantly increased for females at 75 mg/kg over post coitum Days 14 to 17. This was not considered toxicologically relevant since the difference from controls was only slight and/or occurred in the absence of a treatment related distribution.
HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats.
Females at 750 mg/kg had significantly lower neutrophils and significantly increased lymphocyte counts than controls. This was likely secondary to slightly high neutrophil and low lymphocyte means obtained for control animals, and was not considered to be toxicologically relevant.
Minor statistically significant differences arising between controls and animals receiving 250 mg/kg were considered not to represent a change of biological relevance.
CLINICAL CHEMISTRY
At 750 mg/kg the following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from controls:
-Increased alanine aminotransferase; females.
-Increased alkaline phosphatase; both sexes. Also seen for animals at 250 mg/kg, but was significant for males only.
-Reduced total bilirubin; males. Also seen at 75 and 250 mg/kg without dose response.
-Increased glucose; males.
-Lower cholesterol; males. Also seen at 75 and 250 mg/kg.
-Increased inorganic phosphate; males.
MACROSCOPIC EXAMINATION
Macroscopic findings noted for the female at 250 mg/kg that was euthanised in extremis included enlarged mandibular lymph node, thymus reduced in size and GI tract distended with gas.
Findings noted for the female at 750 mg/kg that died due to a gavage error included fluid in the pericardium, perforation of the oesophagus, both adrenals enlarged and many dark red foci on the thymus. In the uterus, 1 foetus and 4 early resorptions were seen in the right uterine horn and 7 foetuses and 1 early resorption were seen in the left uterine horn.
At 750 mg/kg all males and 7/10 females had visibly enlarged livers at the macroscopic examination.
Enlarged liver was also noted for 2 females at 250 mg/kg. This was considered toxicologically relevant.
One female at 250 mg/kg was found with 1 mummified foetus and dark red and watery clear fluid in the uterus. This female did not deliver any live pups.
Incidental findings noted for control and treated animals included enlarged mandibular lymph nodes, yellowish-soft nodule or nodules on the epididymis tail, pelvic dilation of one or both kidneys, enlarged spleen, ectopic splenic tissue, reddish or dark red discoloration of the mandibular or renal lymph nodes, tan focus on the left clitoral gland, yellowish, greenish hard nodule on epididymal adipose tissue, reduced size of the thymus, and alopecia on various body regions. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain and did not show a dose-related incidence trend. As such, these were not considered to be toxicologically relevant.
ORGAN WEIGHTS
At 750 mg/kg terminal body weights were significantly lower for males while significantly increased absolute and relative liver weights (both sexes) were seen. Absolute and relative liver weights were significantly higher for both sexes at 250 mg/kg as well.
Kidney to body weight ratios were significantly higher for males of all treatment groups, but in the absence of any adverse renal findings noted at the microscopic examination, it was not considered to be biologically significant.
Absolute and relative thyroid weights were also significantly higher for males of all treatment groups.
However, the values obtained for controls were slightly low as weights at 75 mg/kg matched the historical mean (mean absolute thyroid weight = 0.029, mean relative thyroid weight = 0.008). As such, the significant differences were not considered to be toxicologically relevant. The lack of thyroid effects for males seen at the histopathological examination also supports this conclusion.
Absolute heart weights were significantly lower for males at 250 mg/kg compared to controls. In the absence of a treatment related distribution, it was not considered to be toxicologically relevant.
MICROSCOPIC EXAMINATION
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test material.
There were treatment-related microscopic findings present in the liver, thyroid gland and kidneys.
Liver
-Centrilobular hypertrophy of the hepatocytes (above background) was present in female rats treated at 250 mg/kg (4/7 rats) up to a slight degree and in male and female rats treated at 750 mg/kg (10/10 and 9/9 rats respectively) up to marked degree.
Thyroid glands
-Hyperplasia and hypertrophy, follicular was present at increased incidence and severity in females treated at 750 mg/kg (5/6) compared to control (1/5), 75 mg/kg (1/5) and 250 mg/kg (0/5) treated rats.
Kidneys
-Hyaline droplets were present at an increased incidence and/or severity in male rats treated at 250 (Group 3; 5/5) and 750 mg/kg (Group 4; 5/5), compared to control (3/5) and 75 mg/kg (Group 2; 5/7) treated males.
The cortical hyaline droplets, representing alpha2µglobulin, were not accompanied by an increase in indicators of tubular damage and the severity grades were within the range seen in untreated male rats.
REPRODUCTIVE PARAMETERS
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.
DEVELOPMENTAL DATA
No toxicologically relevant effects on the gestation index and duration, parturition, maternal care and most aspects of early postnatal pup development (clinical signs, body weight and macroscopy) were observed.
Gestation
The gestation index and duration of gestation were unaffected by treatment up to 750 mg/kg. The gestation index was 100 % for controls and animals at 75 mg/kg, and was 80 % for animals at 250 and 750 mg/kg. There was one female each at 250 and 750 mg/kg with implantation sites only.
Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth and no deficiencies in maternal care were observed.
VIABILITY (OFFSPRING)
Three pups of the control group and five, seven and eleven pups in the 75, 250 and 750 mg/kg groups were found dead or went missing during the first days of lactation. Missing pups were most likely cannibalised. At 250 mg/kg, 5 dead pups came from a single litter. At 750 mg/kg 10 of the 11 dead pups came from two litters, which had 7 and 3 dead pups, respectively. A relationship to treatment could not be excluded at either dose level.
CLINICAL SIGNS (OFFSPRING)
All clinical signs noted for pups were incidental in nature and included scabbing on the hindleg or abdomen, blue spot on the neck or snout, laboured respiration, cold to the touch, pale appearance and lean. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
BODY WEIGHT (OFFSPRING)
Body weights of pups were unaffected by treatment.
GROSS PATHOLOGY (OFFSPRING)
Incidental macroscopic findings of pups that were found dead included no milk in the stomach, insufficient milk in the stomach, partial cannibalism (only the head was present) and/or beginning autolysis. Incidental macroscopic findings noted for surviving pups included scabbing on the right hindleg or abdomen, and blue spot on the neck. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore not considered toxicologically relevant.
OTHER FINDINGS (OFFSPRING)
Mean litter sizes were significantly smaller at 750 mg/kg then compared to controls. A high number of dead pups at first litter check (11, compared to 1 for controls) contributed to this.
The ratio of males/females was significantly lower for litters at 250 and 750 mg/kg. The pup deaths in these groups did not contribute to this as the pup deaths were predominantly of females, and as such this was considered a chance finding.
Table 1 Precoital Time (F0 Generation Postcoitum)
Day of the Pairing Period |
Dose group (mg/kg) |
|||
Control |
75 |
250 |
750 |
|
Number of Females Mated |
||||
1 2 3 4 5 13 |
4 3 2 1 - - |
2 4 4 - - - |
1 5 1 2 1 - |
2 3 3 1 - 1 |
Median Precoital Time Mean Precoital Time N |
2 2.0 10 |
2 2.2 10 |
2 2.7 10 |
3 3.4 10 |
Table 2 Summary of Reproduction Data
|
Dose group (mg/kg) |
|||
Control |
75 |
250 |
750 |
|
Females paired Females mated Females non-mated Pregnant females Non-pregnant females Females with implantation sites only Females with living pups on Day 1 |
10 10 0 10 0 0 10 |
10 10 0 10 0 0 10 |
10 10 0 10 0 1 8 |
10 10 0 10 0 1 8 |
Mating Index (%) Fertility Index (%) Conception Index (%) Gestation Index (%) |
100.0 100.0 100.0 100.0 |
100.0 100.0 100.0 100.0 |
100.0 100.0 100.0 80.0 |
100.0 100.0 100.0 80.0 |
Table 3 Summary of the Number of Corpora Lutea and Implantation Sites at Necropsy
|
|
Dose group (mg/kg) |
|||
Control |
75 |
250 |
750 |
||
Corpora lutea |
Mean SD N |
14.1 1.7 10 |
15.3 3.1 10 |
14.4 1.8 10 |
13.5 2.8 10 |
Implantations |
Mean SD N |
13.0 1.6 10 |
13.0 2.2 10 |
11.3 4.1 10 |
11.7 2.5 10 |
SD = Standard deviation
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 750 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- The key study was conducted in accordance with the standardised guideline OECD 422 under GLP conditions. It was assigned a reliability score of 1 in accordance with the criteria set forth by Klimisch (1997).
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
Effect on fertility: via oral route
A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guidelines OECD 422 and EPA OPPTS 870.3650 under GLP conditions.
Four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test material at 0, 75, 250 and 750 mg/kg/day in propylene glycol. Males were exposed for 28 days (2 weeks prior to mating, during mating, and up to termination) and females were exposed for 41 to 55 days (2 weeks prior to mating, during mating, during postcoitum and during at least 4 days of lactation).
Animals were evaluated for mortality/viability, clinical signs, functional observations and locomotor activity, body weight and food consumption, clinical pathology, macroscopy at termination, organ weights and histopathology on a selection of tissues and reproduction/developmental parameters.
Under the conditions of this study, the NOAEL for parental repeated dose toxicity was determined to be 75 mg/kg bw/day.
No toxicologically relevant effects on reproductive parameters were noted. The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.
Under the conditions of this study, no reproductive toxicity was observed up to the highest dose level tested. The NOAEL for reproductive parameters is therefore considered to be 750 mg/kg/day.
Short description of key information:
TOXICITY TO REPRODUCTION
NOAEL 750 mg/kg/day male and female Wistar Han strain rats.
Justification for selection of Effect on fertility via oral route:
Only one study available.
Effects on developmental toxicity
Description of key information
DEVELOPMENTAL TOXICITY / TERATOGENICITY
NOAEL 75 mg/kg/day Wistar Han strain rats.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 23 July 2012 to 2 October 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EPA OPPTS 870.3650
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Strain: Crl:WI(Han) (outbred, SPF-Quality).
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: The average weight of the males was 326 g and the average weight of the females was 206 g on Day 1 of the study.
- Housing: Pre-mating, animals were housed in groups of 5 animals/sex/cage in plastic cages (height 18 cm). During mating, females were caged together with males on a one-to-one-basis in plastic cages (height 18 cm). Post-mating, males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in plastic cages (height 18 cm). During lactation, pups were kept with the dam until termination in plastic cages (height 18 cm).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet.
- Water (e.g. ad libitum): Water Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24 °C
- Humidity (%): 40 to 70 % relative
- Air changes (per hr): approximately 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark cycle
IN-LIFE DATES: From: 9 August 2012 To: 2 October 2012 - Route of administration:
- oral: gavage
- Vehicle:
- propylene glycol
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS
VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
- Specific gravity: 1.036
- Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenised to a visually acceptable level. No correction was made for the purity of the test material. Adjustment was made for specific gravity of the vehicle. Formulations were placed on a magnetic stirrer during dosing. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The purpose of the analytical phase was to determine the accuracy of preparation, homogeneity and stability of the test material in formulations.
Samples of dose preparations were taken on a single occasion during the treatment phase and were stored and dispatched on dry ice to the test site for formulation analysis.
Formulations were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in the vehicle over 5 hours was also determined (highest and lowest concentration).
Specific sampling instructions:
− Appearance of the formulations was recorded prior to sampling
− Formulations were placed on a magnetic stirrer during sampling
− Sampling was conducted with a clean pipette tip used for every group
− Duplicate samples were taken from each sampling height, with a sample volume of 0.5 mL
− Samples were weighed (grams) on an analytical balance at a precision of 4 decimals
− T, M and B samples were stored on dry ice immediately after sampling
− S (Stability) samples were kept at room temperature under normal laboratory light conditions for
5 hours, and then placed on dry ice
− All samples were transferred to the weighing room at once for dispatch
SAMPLES
In total, 20 samples were included, randomised over 4 formulation groups. Group 1 was the control group. Group 2, 3 and 4 were dosed at 75, 250 and 750 mg/kg bw/day, respectively. The samples of the control group and group 3 were taken in duplicate from the middle position of the container and immediately stored on dry ice.
Samples of treatment groups 2 and 4 were taken in duplicate from the top, middle and bottom position of the container. In addition, stability samples were taken from formulation groups 2 and 4, in duplicate at the middle position of the container, stored for 5 hours at room temperature under normal laboratory light conditions and then placed on dry ice.
The samples were stored at the analytical facility at a target temperature ≤-70 °C.
ANALYTICAL METHOD
The determination was performed using LCMS/MS.
SAMPLE INJECTIONS
The analytical run was acquired in the following order:
- Analytical blank sample
- Calibration curve
- Analytical blank sample
- Analytical blank sample
- Procedural recovery sample low and high (replicate 1)
- Analytical samples
- Stability at storage conditions low and high (replicate 1 and 2)
- Procedural recovery samples low and high (replicate 2)
Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were
analysed by single injection.
ELECTRONIC DATA CAPTURE
System control, data acquisition and data processing were performed using the program Analyst version 1.5.2 (Applied Biosystems, Burlington, Canada).
Electronic raw data was stored in the document management system using KnowledgeTree version 3.5.2c (Raleigh, USA).
FORMULAS
Response (Y)
Peak area test substance [cps]
Calibration curve
Y = bX + a
where:
X = nominal concentration [mg/L]
b = slope [cps × L/mg]
a = intercept [cps]
Analysed concentration (X)
X = ((Y - a) / b) x ((V x d) / w [mg/g]
where:
w = weight sample [mg]
V = volume volumetric flask [mL]
d = dilution factor
(Y - a) / b values are obtained from Analyst
Recovery
(Analysed concentration [mg/g] / Nominal concentration [mg/g]) x 100 [%]
Accuracy
(Analysed concentration [mg/g] / Target concentration [mg/g]) x 100 [%]
Relative difference (relative diff.)
((Ct - C0) / C0) x 100 [%]
where:
Ct = mean concentration of stored samples [mg/g]
C0 = mean concentration of non-stored samples [mg/g]
SPECIFICATIONS
Run Acceptance
Calibration curves with a coefficient of correlation (r) of >0.99 and accuracies of the back calculated values of the calibration standard solutions in the range 85 to 115 % were accepted.
The mean recovery of the procedural recovery samples at each level had to be in the range 90 to 110 %.
The response in all analytical blank samples at the retention time of the test substance should be less than or equal to 30 % compared to the response of the lowest calibration standard (mean response of the duplicate injections).
Acceptance criteria formulations
Preparation of formulations was considered acceptable if the mean accuracy was in the range 90 to 110 % of the target concentration and if the coefficient of variation was ≤ 10 %. Formulations were considered stable if the absolute relative difference between the stored and freshly taken samples was ≤ 10 %.
RESULTS
Calibration curves
Calibration curves were constructed using six concentrations. For each concentration, two responses were acquired. Linear regression analysis was performed using the least squares linear regression with a 1/concentration² weighting factor.
The coefficient of correlation (r) of the calibration curve was 0.9998.
Additionally, there was no response in all analytical blank samples at the retention time of the test material.
Procedural recovery samples
The mean recoveries of the procedural recovery samples fell within the criterion of 90 to 110 %. It demonstrated that the analytical method was adequate for the determination of the test material in the test samples.
Test samples
In the Group 1 formulation, no test material was detected. The concentrations analysed in the formulations of Group 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 90 and 110 %).
The formulations of Group 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10 %). Analysis of Group 2 and Group 4 formulations after storage yielded an absolute relative difference ≤ 10 %. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 5 hours.
Stability at storage conditions
It was observed that the mean accuracy was 94.9 and 98.2 % at the levels high and low, respectively, which is within the criteria set (mean accuracy is in the range 90 to 110 %). It was therefore concluded that the test material was stable in propylene glycol over a storage period of 29 days at a temperature of ≤-70 °C. - Details on mating procedure:
- Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
- Duration of treatment / exposure:
- Males were exposed for 28 days: 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42 to 55 days: 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
- Frequency of treatment:
- Once daily
- Duration of test:
- 28 days for males, 42 to 55 days for females.
- Remarks:
- Doses / Concentrations:
0, 75, 250 and 750 mg/kg
Basis:
actual ingested - No. of animals per sex per dose:
- 10 animals per sex per dose
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Based on the results of a 10-day dose range finding study conducted at the testing laboratory.
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Parental animals were observed for mortality / viability at least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, detailed clinical observations were conducted for all animals after dosing (at no specific time point, but around the same time point for all animals). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded.
BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
FOOD CONSUMPTION: Yes
- Time schedule: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.
FOOD EFFICIENCY: No
WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
OTHER: Observations were carried out for haematology, clotting potential, clinical chemistry and neurobehavioural examinations. - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes. The numbers of former implantation sites and corpora lutea were recorded for all paired females.
- Fetal examinations:
- Each litter was examined to determine the following, if practically possible:
- Mortality/viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were conducted for all animals.
- Bodyweights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation. - Statistics:
- The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to- one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
The following additional methods of statistical analysis were used:
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. - Indices:
- For each group, the following calculations were performed:
- Mating index (%) = (Number of females mated / Number of females paired) x 100
- Fertility index (%) = (Number of pregnant females / Number of females paired) x 100
- Conception index (%) = (Number of pregnant females / Number of females mated) x 100
- Gestation index (%) = (Number of females bearing live pups / Number of pregnant females) x 100
- Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
- Percentage live males at first litter check = (Number of live male pups at first litter check / Number of live pups at first litter check) x 100
- Percentage live females at first litter check = (Number of live female pups at first litter check / Number of live pups at first litter check) x 100
- Percentage of postnatal loss Days 0-4 of lactation = (Number of dead pups on Day 4 of lactation / Number of live pups at first litter check) x 100
- Viability index = (Number of live pups on Day 4 post partum / Number of pups born alive) x 100 - Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
MORTALITY
One female at 250 mg/kg was euthanised in extremis after 26 days of treatment; no definitive cause of death could be determined.
One female at 750 mg/kg was found dead after 33 days of treatment. Macroscopic examination confirmed that her death was secondary to a gavage error, and was in no way related to treatment.
CLINICAL SIGNS
Clinical signs noted for the female at 250 mg/kg that was euthanised in extremis included lethargy, hunched posture, laboured or shallow respiration, rales, piloerection, lean appearance and salivation. These were noted for several days prior to her death.
There were no other toxicology relevant observations noted up to 750 mg/kg.
CLINICAL CHEMISTRY
At 750 mg/kg the following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from controls:
-Increased alanine aminotransferase
-Increased alkaline phosphatase
MACROSCOPIC EXAMINATION
Macroscopic findings noted for the female at 250 mg/kg that was euthanised in extremis included enlarged mandibular lymph node, thymus reduced in size and GI tract distended with gas.
Findings noted for the female at 750 mg/kg that died due to a gavage error included fluid in the pericardium, perforation of the oesophagus, both adrenals enlarged and many dark red foci on the thymus. In the uterus, 1 foetus and 4 early resorptions were seen in the right uterine horn and 7 foetuses and 1 early resorption were seen in the left uterine horn.
At 750 mg/kg 7/10 females had visibly enlarged livers at the macroscopic examination.
Enlarged liver was also noted for 2 females at 250 mg/kg. This was considered toxicologically relevant.
One female at 250 mg/kg was found with 1 mummified foetus and dark red and watery clear fluid in the uterus. This female did not deliver any live pups.
Incidental findings noted for control and treated animals included enlarged mandibular lymph nodes, pelvic dilation of one or both kidneys, enlarged spleen, ectopic splenic tissue, reddish or dark red discoloration of the mandibular or renal lymph nodes, tan focus on the left clitoral gland, reduced size of the thymus, and alopecia on various body regions. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain and did not show a dose-related incidence trend. As such, these were not considered to be toxicologically relevant.
ORGAN WEIGHTS
At 750 mg/kg significantly increased absolute and relative liver weights (both sexes) were seen. Absolute and relative liver weights were significantly higher at 250 mg/kg as well.
MICROSCOPIC EXAMINATION
There were no morphological findings in the reproductive organs which could be attributed to the test material.
There were treatment-related microscopic findings present in the liver and thyroid gland.
Liver
-Centrilobular hypertrophy of the hepatocytes (above background) was present in female rats treated at 250 mg/kg (4/7 rats) up to a slight degree and in female rats treated at 750 mg/kg (9/9 rats) up to marked degree.
Thyroid glands
-Hyperplasia and hypertrophy, follicular was present at increased incidence and severity in females treated at 750 mg/kg (5/6) compared to control (1/5), 75 mg/kg (1/5) and 250 mg/kg (0/5) treated rats.
REPRODUCTIVE PARAMETERS
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.
DEVELOPMENTAL DATA
No toxicologically relevant effects on the gestation index and duration, parturition, maternal care and most aspects of early postnatal pup development (clinical signs, body weight and macroscopy) were observed.
Gestation
The gestation index and duration of gestation were unaffected by treatment up to 750 mg/kg. The gestation index was 100 % for controls and animals at 75 mg/kg, and was 80 % for animals at 250 and 750 mg/kg. There was one female each at 250 and 750 mg/kg with implantation sites only.
Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth and no deficiencies in maternal care were observed. - Dose descriptor:
- NOAEL
- Effect level:
- 75 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 75 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
The postnatal loss and viability index were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.
VIABILITY (OFFSPRING)
Three pups of the control group and five, seven and eleven pups in the 75, 250 and 750 mg/kg groups were found dead or went missing during the first days of lactation. Missing pups were most likely cannibalised. At 250 mg/kg, 5 dead pups came from a single litter. At 750 mg/kg 10 of the 11 dead pups came from two litters, which had 7 and 3 dead pups, respectively. A relationship to treatment could not be excluded at either dose level.
CLINICAL SIGNS (OFFSPRING)
All clinical signs noted for pups were incidental in nature and included scabbing on the hindleg or abdomen, blue spot on the neck or snout, laboured respiration, cold to the touch, pale appearance and lean. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
BODY WEIGHT (OFFSPRING)
Body weights of pups were unaffected by treatment.
GROSS PATHOLOGY (OFFSPRING)
Incidental macroscopic findings of pups that were found dead included no milk in the stomach, insufficient milk in the stomach, partial cannibalism (only the head was present) and/or beginning autolysis. Incidental macroscopic findings noted for surviving pups included scabbing on the right hindleg or abdomen, and blue spot on the neck. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore not considered toxicologically relevant.
OTHER FINDINGS (OFFSPRING)
Mean litter sizes were significantly smaller at 750 mg/kg then compared to controls. A high number of dead pups at first litter check (11, compared to 1 for controls) contributed to this.
The ratio of males/females was significantly lower for litters at 250 and 750 mg/kg. The pup deaths in these groups did not contribute to this as the pup deaths were predominantly of females, and as such this was considered a chance finding. - Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- Under the conditions of this study, the NOAEL for developmental parameters is considered to be 75 mg/kg/day. The test material therefore requires classification in accordance with EU criteria for developmental toxicity as Category 2.
- Executive summary:
A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guidelines OECD 422 and EPA OPPTS 870.3650 under GLP conditions.
Four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test material at 0, 75, 250 and 750 mg/kg/day in propylene glycol. Males were exposed for 28 days (2 weeks prior to mating, during mating, and up to termination) and females were exposed for 41 to 55 days (2 weeks prior to mating, during mating, during postcoitum and during at least 4 days of lactation).
Animals were evaluated for mortality/viability, clinical signs, functional observations and locomotor activity, body weight and food consumption, clinical pathology, macroscopy at termination, organ weights and histopathology on a selection of tissues and reproduction/developmental parameters.
Under the conditions of this study, the NOAEL for parental repeated dose toxicity was determined to be 75 mg/kg bw/day.
The mean litter size was smaller at 750 mg/kg than controls and higher pup mortality was evident at both 250 and 750 mg/kg. No toxicologically significant changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and clinical signs, bodyweights and macroscopy of pups).
Under the conditions of this study, the NOAEL for developmental parameters is considered to be 75 mg/kg/day. The test material therefore requires classification in accordance with EU criteria for developmental toxicity as Category 2.
Reference
Table 1 Summary of Developmental Data (F0 - Generation - Lactation)
|
Dose group (mg/kg) |
|||
Control |
75 |
250 |
750 |
|
Total number of litters |
10 |
10 |
8 |
8 |
Duration of gestation Mean* SD N |
21.2 0.4 10 |
21.2 0.4 10 |
21.4 0.5 8 |
21.4 0.5 8 |
Dead pups at first litter check Litters affected†Total Mean* SD N |
1 1 0.1 0.3 10 |
1 1 0.1 0.3 10 |
2 4 0.5 1.1 8 |
3 11 1.4 2.5 8 |
Living pups at first litter check % of males/females†Total Mean* SD N |
55/45 119 11.9 2.4 10 |
48/53 120 12.0 2.4 10 |
38/62†89 11.1 2.6 8 |
39/61†75 9.4* 1.8 8 |
Postnatal loss % of living pups Litters affected†Total†Mean* SD N |
1.7 2 2 0.2 0.4 10 |
3.3 2 4 0.4 0.8 10 |
3.4 2 3 0.4 0.7 8 |
0.0 0 0 0.0 0.0 8 |
Viability index†|
98.3 |
96.7 |
96.6 |
100.0 |
*Significant at 5 % using the Steel-test
†Significant at 5 % using Fisher’s Exact test
SD = Standard deviation
Table 2 Summary of Pup Bodyweights in g (F0 - Generation - Lactation)
Day |
Sex |
|
Dose group (mg/kg) |
|||
Control |
75 |
250 |
750 |
|||
1 |
Male |
Mean SD N |
6.0 0.3 10 |
5.9 0.8 10 |
5.9 0.6 8 |
5.9 0.8 8 |
Female |
Mean SD N |
5.6 0.4 10 |
5.7 0.7 10 |
5.8 0.6 8 |
5.6 0.8 8 |
|
Male + Female |
Mean SD N |
5.8 0.3 10 |
5.8 0.7 10 |
5.9 0.6 8 |
5.7 0.8 8 |
|
4
|
Male |
Mean SD N |
8.9 0.6 10 |
8.9 1.4 10 |
8.9 1.2 8 |
9.3 1.4 8 |
Female |
Mean SD N |
8.6 0.9 10 |
8.6 1.2 10 |
8.7 1.2 8 |
8.6 1.4 8 |
|
Male + Female |
Mean SD N |
8.8 0.8 10 |
8.8 1.3 10 |
8.8 1.2 8 |
8.9 1.5 8 |
SD = Standard deviation
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 75 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- The key study was conducted in accordance with the standardised guideline OECD 422 under GLP conditions. It was assigned a reliability score of 1 in accordance with the criteria set forth by Klimisch (1997).
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Additional information
Effect on developmental toxicity: via oral route
A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guidelines OECD 422 and EPA OPPTS 870.3650 under GLP conditions.
Four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test material at 0, 75, 250 and 750 mg/kg/day in propylene glycol. Males were exposed for 28 days (2 weeks prior to mating, during mating, and up to termination) and females were exposed for 41 to 55 days (2 weeks prior to mating, during mating, during postcoitum and during at least 4 days of lactation).
Animals were evaluated for mortality/viability, clinical signs, functional observations and locomotor activity, body weight and food consumption, clinical pathology, macroscopy at termination, organ weights and histopathology on a selection of tissues and reproduction/developmental parameters.
Under the conditions of this study, the NOAEL for parental repeated dose toxicity was determined to be 75 mg/kg bw/day.
The mean litter size was smaller at 750 mg/kg than controls and higher pup mortality was evident at both 250 and 750 mg/kg. No toxicologically significant changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and clinical signs, bodyweights and macroscopy of pups).
Under the conditions of this study, the NOAEL for developmental parameters is considered to be 75 mg/kg/day. The test material therefore requires classification in accordance with EU criteria for developmental toxicity as Category 2.
Justification for selection of Effect on developmental toxicity: via oral route:
Only one study available.
Justification for classification or non-classification
In accordance with the criteria for classification as defined in Annex I, Regulation (EC) 1272/2008, the test material requires classification for developmental toxicity as Category 2.
Additional information
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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