Registration Dossier

Administrative data

Description of key information

A subchronic 90-day oral toxicity study with rats revealed a NOAEL of 1000 mg/kg bw/day (limit dose). In this study, no adverse changes in clinical appearance, body weights, food consumption, ophthalmoscopic findings, clinical laboratory investigations, macroscopic findings, organ weights and microscopic findings that were considered to be an effect of treatment were observed.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 August 2011 to 24 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
- ANIMAL HOUSING
Upon arrival, all animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The animal facilities at WIL Research are accredited by AAALAC International. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.
- DIET, DRINKING WATER, AND MAINTENANCE
The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal), is a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research. Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the study, except during the period of fasting prior to clinical pathology blood collection when food, but not water, was withheld. Municipal water supplying the facility was analyzed for contaminants according to SOPs. The results of the diet and water analyses are maintained at WIL Research. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.
- ENVIRONMENTAL CONDITIONS
All animals were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and humidity controls were set to maintain environmental conditions of 71 ± 5°F (22 ± 3°C) and 50 ± 20%, respectively. Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. Actual mean daily temperature ranged from 69.9°F to 70.7°F (21.1°C to 21.5°C) and mean daily relative humidity ranged from 38.4% to 54.1% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. The 12-hour light/12-hour dark photoperiod was interrupted as necessary to allow for the performance of protocol-specified activities. Air handling units were set to provide a minimum of 10 fresh air changes per hour.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The vehicle and test item formulations were administered orally by gavage via an appropriately sized flexible Teflon®-shafted, stainless steel ball-tipped dosing cannula once daily for 91 or 92 consecutive days, through the day prior to the primary necropsy. The dose volume for all groups was 10 mL/kg. Individual doses were based on the most recently recorded body weights to provide the correct mg/kg/day dosage. Adjusted doses became effective the day of collection of the weekly body weights. The first day of dosing was study day 0; the first week of dosing was study week 0.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test item at concentrations of 50 to 100 mg/mL was previously confirmed following 10 days of refrigerated storage (approximately 2°C to 8°C). Prior to the initiation of dose administration, samples for homogeneity determination were collected from the top, middle, and bottom strata of the 25, 50, and 100 mg/mL dosing formulations. Aliquots approximately equivalent in volume to a daily dispensation aliquot were then transferred from the pre-initiation batch into the same type of storage container that was used for the daily aliquots for dosing and stored refrigerated (approximately 2°C to 8°C) for 10 days. Following the storage period, each aliquot was resuspended for a minimum of 30 minutes, and samples were collected from the top and bottom strata of each aliquot for assessment of 10-day refrigerated stability and aliquot resuspension homogeneity. Samples for concentration analysis were collected during study weeks 0, 3, 7, and 12 from the middle stratum of each dosing formulation (including the control group).
One duplicate set of samples was analyzed and the remaining duplicate set was refrigerated (approximately 2°C to 8°C) and retained as backup samples until the results were verified as acceptable. All analyses were conducted by the WIL Research Analytical Chemistry Department utilizing a validated gas chromatography method using flame ionization detection.
Duration of treatment / exposure:
91 or 92 days and 28 day recovery period
Frequency of treatment:
1 per day
Remarks:
Doses / Concentrations:
0 mg/ kg bw/ day
Basis:
other: gavage
Remarks:
Doses / Concentrations:
250 mg/kg bw/ day
Basis:
other: gavage
Remarks:
Doses / Concentrations:
500 mg/kg bw/ day
Basis:
other: gavage
Remarks:
Doses / Concentrations:
1000 mg/kg bw/ day
Basis:
other: gavage
No. of animals per sex per dose:
Control group: 15 males, 15 females
250 mg/kg bw/d: 10 males, 10 females
500 mg/kg bw/d: 10 males, 10 females
1000 mg/kg bw/d: 15 males, 15 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The highest dose of 1000 mg/kg body weight (bw)/day was selected on the basis of a 4-week oral toxicity study in rats, conducted according to OECD Test Guideline 407 (Strobel, 1995). In the referenced study, a no-observed-effect level (NOEL) of 1000 mg/kg bw/day was established. For studies performed according to OECD Guideline 408, 1000 mg/kg/day is a common limit dosage level. The lower doses of 500 and 250 mg/kg bw/day were chosen due to the recommendation on OECD Test Guideline 408 that spacing factors of 2-4 should be used for dose selection. The selected route of administration for this study was oral (gavage) because this is a potential route of exposure for humans and the requirement for the study was for chemical registration purposes and not specifically with regard to cosmetic use.

- Rationale for animal assignment: Randomization procedure based on body weight

- Post-exposure recovery period in satellite groups: 28 d
Positive control:
none
Observations and examinations performed and frequency:
SURVIVAL: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were performed twice daily during the dosing period, at the time of dose administration and approximately 1 to 2 hours following dose administration. During the recovery period, the animals were observed once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded approximately weekly beginning during acclimation and at the time of randomization. Body weights were also recorded on the day prior to the first day of the scheduled necropsies. During study weeks 12 to 13, body weights were recorded twice weekly.

FOOD CONSUMPTION:
- Individual food consumption was recorded approximately weekly beginning during acclimation and throughout the study. During study weeks 12 and 13, food consumption was recorded twice weekly.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ocular examinations were conducted on all animals prior to randomization (study week -2), near the end of the treatment period (study week 12), and near the end of the recovery period (study week 16).
- Dose groups that were examined: all dose groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: time of the primary and recovery necropsies for those animals scheduled for necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight prior to blood collection
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: time of the primary and recovery necropsies for those animals scheduled for necropsy
- Animals fasted: Yes, overnight prior to blood collection
- How many animals: all animals
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: time of the primary and recovery necropsies for those animals scheduled for necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: study week 12
- Dose groups that were examined: 10 animals/sex/group
- Battery of functions tested: home cage observations/ handling observations/ open field observations/ sensory activity /neuromuscular observations/physiological observations/ locomotor activity

DETERMINATION OF ESTROUS CYCLES: Yes
- Time schedule for examinations: Daily vaginal lavages were performed on all females to determine the stage of estrus beginning 2 weeks prior to and on the day of the scheduled primary necropsy. Vaginal lavage samples were microscopically evaluated to determine the stage of estrus.
- Dose groups that were examined: all females.

PLASMA BIOANALYSIS: Yes
- Time schedule for examinations: 6 hours following dosing administration on study days 7, 28, and 84.
- Dose groups that were examined: 4 animals/sex/group
Sacrifice and pathology:
ANATOMIC PATHOLOGY
- A complete necropsy was conducted on all animals. Animals were euthanized by carbon dioxide inhalation followed by exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.
- Organ weight
- Slide preparation and microscopic examination

SPERMATOGENIC EVALUATIONS
- Dose group: all males (10/group) at the primary necropsy
- Motility/ Viability assessment
- Morphology assessment
- Enumeration of epididymal and testicular sperm, numbers and sperm production rate
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test item-treated group with the control group by sex.
Body weight, body weight change, food consumption, continuous FOB, clinical pathology (except gamma glutamyltransferase [GGT]), estrous length, sperm production rate, epididymal and testicular sperm numbers, and organ weight data were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test item-treated groups with the control group. Functional observational battery parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test (Steel and Torrie, 1980). The percentage of motile spermatozoa and the percentage of sperm with normal
morphology were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed significance (p<0.05), Dunn’s test (Dunn, 1964) was used to compare to the test item-treated groups with the control group. GGT values under range were assigned a value of 0.1 (half the lower limit of quantitation) for statistical analysis and reporting. GGT data were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed significance (p< 0.05), Dunn’s test (Dunn, 1964) was used to compare the test item-treated groups with the control group.
Clinical signs:
no effects observed
Description (incidence and severity):
One female rat in the control group was found dead on study day 84 subsequent to mechanical trauma. All other animals survived to the scheduled necropsies.
Mortality:
no mortality observed
Description (incidence):
One female rat in the control group was found dead on study day 84 subsequent to mechanical trauma. All other animals survived to the scheduled necropsies.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights were unaffected by test item administration. There were no statistically significant differences when the control and test item-treated groups were compared.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
not applicable
Food efficiency:
no effects observed
Description (incidence and severity):
Food consumption was unaffected by test item administration. Any statistically significant differences in food consumption were not test item-related.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmic lesions indicative of toxicity were observed in any of the test item-treated groups.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test item-related alterations in hematology and coagulation parameters at the primary or recovery necropsy.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test item-related effects on serum chemistry parameters.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test item-related effects on urinalysis parameters.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Neurobehaviour patterns were unaffected by the test item administration. There were no statistically significant differences when the test item-treated males and females were compared with the control group at the study week 12 evaluation.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher liver and kidney weights were noted in the 1000 mg/kg/day group but considered non adverse.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related gross observations were noted at the primary or recovery necropsy.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted at the primary necropsy in the livers of the 250, 500, and 1000 mg/kg/day group females but considered non adverse.
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL CHEMISTRY:
Slightly higher test item-related mean calcium values were noted in the 500 and 1000 mg/kg/day group males at the primary necropsy. The relatively small difference in the mean values between the test item-treated groups and the control group at the primary necropsy was therefore not considered an adverse finding.
The mean alanine aminotransferase (ALT) value for the 1000 mg/kg/day group males was higher than the mean ALT value for the control group at the recovery necropsy, but was within the range of means (35 to 59 U/L) recorded in the historical control database. Because the observed mean value was within the range of means in the WIL Research historical control database and no other serum chemistry values associated with liver disease were elevated, this finding was considered due to biological variation. The mean serum alkaline phosphatase (ALP) value for the 1000 mg/kg/day group females was lower than the mean ALP value for the control group at the recovery necropsy but was not considered test item. Higher ALP values may be associated with liver disease, but lower values may be indicative of primary or secondary malnutrition (Lum, 1995). In this study, however, all mean serum ALP values of control and test item-treated animals were within the mean values (37 to 69 U/L) recorded in the WIL Research historical control database suggesting that the low values were due to biological variation and were not test item-related.

URINALYSIS:
A statistically significantly higher mean specific gravity value was noted in the 1000 mg/kg/day group males at the primary necropsy and a lower mean pH value was noted in the 1000 mg/kg/day group males at the primary necropsy. These differences were not considered to be test item-related as both findings were within the range of means (5.9 to 8.6 for pH; 1.0206 to 1.0633 for specific gravity) recorded in the WIL Research historical control database, there was not a statistically significant difference between control and test item-treated groups at the recovery necropsy, and no correlating histological changes were noted in the kidneys.

ORGAN WEIGHTS:
Higher mean liver and kidney weights (absolute and relative to body and brain weights) were noted in the 1000 mg/kg/day group females at the primary necropsy and higher mean liver weight relative to final body weight was noted in the 1000 mg/kg/day group males at the primary and recovery necropsies. These differences were relatively small, i.e., not more than 18% as compared with the control group, and were not detected at the recovery necropsy indicating that the organ weight effects were nonadverse.

GROSS PATHOLOGY:
Dilated renal pelvis was observed in the 250 and 500 mg/kg/day group males at the primary necropsy, and in the 1000 mg/kg/day group males at the recovery necropsy, but this finding was unilateral and occurred only in males, and was therefore not considered to be test item-related. A renal mass was noted in one 1000 mg/kg/day group female at the recovery necropsy.

HISTOPATHOLOGY:
Mild vacuolation of hepatocytes was noted in one 1000 mg/kg/day group male and in 1 control group male, indicating that this finding can be a normal background lesion and was therefore not test item-related in the males. However in the females at the primary necropsy, minimal to mild microvesicular vacuolation of hepatocytes was noted in the females in the 250 and 500 mg/kg/day groups (3 and 4 animals, respectively) and 3 of 10 animals in the 1000 mg/kg/day group had mild vacuolation of hepatocytes which for 2 of these animals was further specified as microvesicular hepatocellular vacuolation, suggesting a relationship with the test item. No instances of hepatocellular vacuolation were noted in the control or 1000 mg/kg/day group females examined at the recovery necropsy. Taking into account the mild nature of the vacuolation at the primary necropsy, the lack of associated liver enzyme alterations, the lack of persistence of the vacuolation at the recovery necropsy, and historical data, the hepatocellular vacuolation, mainly evident as microvesicular hepatocellular vacuolation, was considered to be an adaptive change and nonadverse.
One female in the 1000 mg/kg/day group at the recovery necropsy had a renal carcinoma, but it was not considered test item-related as there were no other lesions associated with renal tumor progression and no evidence of renal toxicity in any other animal. Spontaneous renal tubule carcinomas in rats have been previously described and were considered most likely attributable to genetic predisposition.

ESTROUS CYCLE:
Estrous cycles were unaffected by test item administration. There were no statistically significant differences when the test item-treated females were compared to the control group.

SPERMATOGENIC EVALUATIONS:
There were no test item-related effects on spermatogenic endpoints (testicular and epididymal sperm numbers, sperm production rate, motility, and the percentage of morphologically normal sperm) observed at any dosage level.

PLASMA BIOANALYSIS AND EXPOSURE ASSESSMENT
Plasma concentrations of the test item and the two metabolites ranged from the lower limit of quantitation (LLQ) to 41,700 ng/mL (178.7 μM) for the test item, from the LLQ to 16,600 ng/mL (80.9 μM) for metabolite 1, and from 198 ng/mL to 21,600 ng/mL (98.5 μM) for metabolite 2. The mean plasma concentrations for all 3 analytes increased with dosage on all 3 sampling days in both genders. In females the mean the test item plasma concentrations were generally higher than the mean male value of the corresponding group and day of sampling. In contrast, the mean values for the 2 metabolites were lower in females than the corresponding male values. Regarding the sum of the three analytes (the test item and metabolites) a dose-dependent increase in analyte concentrations was visible. For both genders a plasma level of the combined analytes of approximately 50, 100, and 200 μM was observed respectively at a daily dosage of 250, 500, and 1000 mg/kg body weight, both on days 7 and 28. These data underline the linear dose dependence of plasma levels which is not evident from looking at the the test item data alone due to its rapid metabolism. There was a slight decline of combined plasma levels on study day 84, which was more apparent in males than in females; this may indicate some sort of adaption of the animals to the prolonged test item administration. In the control animals, the test item and metabolite 1 plasma levels were below the lower limit of quantification (50 ng/mL, equivalent to 0.21 to 0.24 μM for the test item and metabolite 1, respectively). Metabolite 2 was detected in control animals at mean levels of 1.1 μM to 2.8 μM, which is consistent with its occurrence as an endogenous metabolite. In summary, plasma data showed dose dependence of the test item absorption, metabolism and no indication of bioaccumulation over time.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Critical effects observed:
not specified
Conclusions:
Based on the results of this study, oral administration of the test item to Crl:CD(SD) rats over a period of 91 or 92 days resulted in a no-observed-adverse-effect level (NOAEL) of 1000 mg/kg/day, the highest dosage level investigated.
Executive summary:

Objective:

The objective of the study was to evaluate the potential toxicity and toxicokinetic profile of the test item when administered orally, by gavage, to Sprague Dawley rats for a minimum of 91 days. In addition, a 28-day recovery period assessed the reversibility, persistence, or delayed occurrence of potential test item-related effects. The protocol was designed to be in general accordance with the OECD Guidelines for the Testing of Chemicals (Guideline 408). The study was conducted in accordance with appropriate GLP guidelines except for the evaluation report of the bioanalytical plasma data that was written by the Sponsor.

 

Study design:

The test item in the vehicle (deionized water) was administered orally by gavage once daily for a minimum of 91 consecutive days to 3 groups (Groups 2-4) of Crl:CD(SD) rats. Dosage levels were 250, 500, and 1000 mg/kg/day for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) received the vehicle (deionized water) on a comparable regimen. The dose volume was 10 mL/kg for all groups. Groups 1 and 4 each consisted of 15 animals/sex and Groups 2 and 3 each consisted of 10 animals/sex. Following approximately 13 weeks of dose administration, 10 rats/sex/group were euthanized; the remaining ≤ 5 rats/sex in the control and high-dose groups were euthanized following a 28-day nondosing (recovery) period. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed approximately weekly. Individual body weights and food consumption were recorded approximately weekly. Functional observational battery (FOB) and locomotor activity data were recorded for all animals prior to the initiation of dose administration, and for 10 animals/sex/group during study week 12. Ophthalmic examinations were performed during study weeks -2, 12, and 16. Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for animals at the primary (study week 13) and recovery (study week 17) necropsies. Daily vaginal lavages for estrous cycle determination were performed on all females beginning 2 weeks prior to the primary necropsy. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically. Microscopic examination was performed on a wide range of listed tissues from the animal found dead and all animals in the control and 1000 mg/kg/day groups at the scheduled primary necropsy. The liver was examined from all females in the 250 and 500 mg/kg/day groups at the primary necropsy and from all females in the control and 1000 mg/kg/day groups at the recovery necropsy. Gross lesions were examined from all animals at the primary and recovery necropsies. Spermatogenic endpoints were evaluated for all males at the primary necropsy.

For plasma bioanalysis, blood samples were collected from 4 animals/sex/group at approximately 6 hours after dose administration on study days 7, 28, and 84.

 

Results:

There were no test item-related effects on survival, body weight, food consumption, FOB or locomotor activity assessment, hematology, coagulation, urinalysis, ophthalmic examinations, estrous cycle, macroscopic examinations, or spermatogenic endpoints. Yellow staining on the ventral trunk and/or around the anogenital and/or urogenital areas was noted in the 1000 mg/kg/day group females beginning on study day 11 and sporadically throughout the dosing period.

Nonadverse test item-related higher calcium values were noted in the 500 and 1000 mg/kg/day group males at the primary necropsy. Nonadverse test item-related higher mean liver and kidney weights (absolute and relative to body and brain weights) were noted in the 1000 mg/kg/day group females at the primary necropsy and higher mean liver weight relative to final body weight was noted in the 1000 mg/kg/day group males at the primary and recovery necropsies. In the females from the 1000 mg/kg/day group at the primary necropsy, 3 of 10 animals had mild vacuolation of hepatocytes, which for 2 of these animals was further specified as microvesicular hepatocellular vacuolation. From the extension of histological evaluations, minimal to mild microvesicular hepatocellular vacuolation was also noted at the primary necropsy in 3 of 10 females in the 250 mg/kg/day group and in 4 of 10 females in the 500 mg/kg/day group. No instances of vacuolation were noted in the control or 1000 mg/kg/day group females examined at the recovery necropsy. Taking into account the mild nature of the vacuolation at the primary necropsy, the lack of associated liver enzyme alterations, the lack of persistence of the vacuolation at the recovery necropsy, and historical data, the hepatocellular vacuolation, mainly evident as microvesicular hepatocellular vacuolation, was considered to be an adaptive change and nonadverse.

Plasma concentrations of the test item and the two metabolites ranged from the lower limit of quantitation (LLQ) to 41,700 ng/mL (178.7μM) for the test item, from the LLQ to 16,600 ng/mL (80.9μM) for metabolite 1, and from 198 ng/mL to 21,600 ng/mL (1.2 - 98.5μM) for metabolite 2. The mean plasma concentrations for all 3 analytes increased with dosage on all 3 sampling days in both genders. In females the mean test item plasma concentrations were generally higher than the mean male value of the corresponding group and day of sampling. In contrast, the mean values for the 2 metabolites were lower in females than the corresponding male values. Regarding the sum of the 3 analytes (the test item, 2 metabolites) a dose-dependent increase in analyte concentrations was visible. For both genders a plasma level of the combined analytes of approximately 50, 100, and 200μM was observed respectively at a daily dosage of 250, 500, and 1000 mg/kg body weight, both on days 7 and 28. These data underline the linear dose dependence of plasma levels which are not evident from looking at the test item data alone due to its rapid metabolism. There was a slight decline of combined plasma levels on study day 84, which was more apparent in males than in females; this may indicate some sort of adaptation of the animals to the prolonged test item administration. In summary, plasma data showed dose dependence of test item absorption, metabolism and no indication of bioaccumulation over time.  

Conclusion:

Based on the results of this study, oral administration of the test item to Crl:CD(SD) rats over a period of 91 or 92 days resulted in a No-Observed-Adverse-Eeffect Level (NOAEL) of 1000 mg/kg/day, the highest dosage level investigated.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP and guideline study without restrictions

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Additional information

Oral:

The potential toxicity and toxicokinetic profile of the test item was evaluated in a repeated dose 90- day oral toxicity study in Sprague Dawley rats. In addition, a 28-day recovery period assessed the reversibility, persistence, or delayed occurrence of potential test item-related effects. The study was designed to be in compliance with the GLP regulation and the OECD Guidelines for the Testing of Chemicals (Guideline 408).

The test item in the vehicle (deionized water) was administered orally by gavage once daily for a minimum of 91 consecutive days to 3 groups (Groups 2-4) of Crl:CD(SD) rats. Dosage levels were 250, 500, and 1000 mg/kg/day for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) received the vehicle (deionized water) on a comparable regimen. The dose volume was 10 mL/kg for all groups. Groups 1 and 4 each consisted of 15 animals/sex and Groups 2 and 3 each consisted of 10 animals/sex. Following approximately 13 weeks of dose administration, 10 rats/sex/group were euthanized; the remaining ≤ 5 rats/sex in the control and high-dose groups were euthanized following a 28-day nondosing (recovery) period. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed approximately weekly. Individual body weights and food consumption were recorded approximately weekly. Functional observational battery (FOB) and locomotor activity data were recorded for all animals prior to the initiation of dose administration, and for 10 animals/sex/group during study week 12. Ophthalmic examinations were performed during study weeks -2, 12, and 16. Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for animals at the primary (study week 13) and recovery (study week 17) necropsies. Daily vaginal lavages for estrous cycle determination were performed on all females beginning 2 weeks prior to the primary necropsy. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically. Microscopic examination was performed on a wide range of listed tissues from the animal found dead and all animals in the control and 1000 mg/kg/day groups at the scheduled primary necropsy. The liver was examined from all females in the 250 and 500 mg/kg/day groups at the primary necropsy and from all females in the control and 1000 mg/kg/day groups at the recovery necropsy. Gross lesions were examined from all animals at the primary and recovery necropsies. Spermatogenic endpoints were evaluated for all males at the primary necropsy.

For plasma bioanalysis, blood samples were collected from 4 animals/sex/group at approximately 6 hours after dose administration on study days 7, 28, and 84.

There were no test item-related effects on survival, body weight, food consumption, FOB or locomotor activity assessment, hematology, coagulation, urinalysis, ophthalmic examinations, estrous cycle, macroscopic examinations, or spermatogenic endpoints. Yellow staining on the ventral trunk and/or around the anogenital and/or urogenital areas was noted in the 1000 mg/kg/day group females beginning on study day 11 and sporadically throughout the dosing period.

Nonadverse test item-related higher calcium values were noted in the 500 and 1000 mg/kg/day group males at the primary necropsy. Nonadverse test item-related higher mean liver and kidney weights (absolute and relative to body and brain weights) were noted in the 1000 mg/kg/day group females at the primary necropsy and higher mean liver weight relative to final body weight was noted in the 1000 mg/kg/day group males at the primary and recovery necropsies. In the females from the 1000 mg/kg/day group at the primary necropsy, 3 of 10 animals had mild vacuolation of hepatocytes, which for 2 of these animals was further specified as microvesicular hepatocellular vacuolation. From the extension of histological evaluations, minimal to mild microvesicular hepatocellular vacuolation was also noted at the primary necropsy in 3 of 10 females in the 250 mg/kg/day group and in 4 of 10 females in the 500 mg/kg/day group. No instances of vacuolation were noted in the control or 1000 mg/kg/day group females examined at the recovery necropsy. Taking into account the mild nature of the vacuolation at the primary necropsy, the lack of associated liver enzyme alterations, the lack of persistence of the vacuolation at the recovery necropsy, and historical data, the hepatocellular vacuolation, mainly evident as microvesicular hepatocellular vacuolation, was considered to be an adaptive change and nonadverse.

Plasma concentrations of the test item and the two main metabolites ranged from the lower limit of quantitation (LLQ) to 41,700 ng/mL (178.7μM) for the test item, from the LLQ to 16,600 ng/mL (80.9μM) for metabolite 1, and from 198 ng/mL to 21,600 ng/mL (1.2 - 98.5μM) for metabolite 2. The mean plasma concentrations for all 3 analytes increased with dosage on all 3 sampling days in both genders. In females the mean the test item plasma concentrations were generally higher than the mean male value of the corresponding group and day of sampling. In contrast, the mean values for the 2 metabolites were lower in females than the corresponding male values. Regarding the sum of the 3 analytes (the test item, 2 metabolites) a dose-dependent increase in analyte concentrations was visible. For both genders a plasma level of the combined analytes of approximately 50, 100, and 200μM was observed respectively at a daily dosage of 250, 500, and 1000 mg/kg body weight, both on days 7 and 28. These data underline the linear dose dependence of plasma levels which are not evident from looking at the test item data alone due to its rapid metabolism. There was a slight decline of combined plasma levels on study day 84, which was more apparent in males than in females; this may indicate some sort of adaptation of the animals to the prolonged the test item administration. In summary, plasma data showed dose dependence of the test item absorption, metabolism and no indication of bioaccumulation over time.

 

In addition the test item was investigated in a 28-day subacute oral toxicity study in rats. The dose levels for the toxicity study were selected to be 0, 50, 200 and 1000 mg/kg bw/day. The test substance was administered daily for 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females. The following parameters were evaluated: clinical signs daily, body weight and food consumption weekly; ophthalmoscopy at week 4; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues. There were no changes in clinical appearance, body weights, food consumption, ophthalmoscopic findings, clinical laboratory investigations, macroscopic findings, organ weights and microscopic findings that were considered to be an effect of treatment.

 

Based on the results of the key study, oral administration of the test item to Crl:CD(SD) rats over a period of 91 or 92 days resulted in a No-Observed-Adverse-Effect Level (NOAEL) of 1000 mg/kg/day, the highest dosage level investigated.

 

Inhalation:

No repeated dose inhalation toxicity study was performed as the vapour pressure (0.44 Pa) is low and the inhalatory route is not considered important. Also the use in mainly closed systems when producing the substance and the final products as hair and nail cosmetics, indicate that the inhalation exposure is not relevant.

 

Dermal:

In accordance with column 2 of REACH Annex IX, a dermal repeated dose toxicity study (required in section 8.6.2) does not need to be conducted as the available short term toxicity studies (oral and dermal, see sections 7.2.1 and 7.2.3), as well as the available oral repeated dose toxicity studies (see section 7.5.1) together with the low log Po/w (<0) indicate a low dermal toxicity after repeated application.


Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
No study on sub-chronic toxicity (by inhalation) was performed, as vapour pressure (0.44 Pa) is low and the inhalation route is considered of low relevance. Production is limited to closed systems only and use of product as hair and nail cosmetic, indicates that inhalation exposure is not relevant.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
No study on sub-chronic toxicity (by inhalation) was performed, as vapour pressure (0.44 Pa) is low and the inhalation route is considered of low relevance. Production is limited to closed systems only and use of product as hair and nail cosmetic, indicates that inhalation exposure is not relevant.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
In accordance with 1907/2006/EC Annex IX (section 8.6.2), a sub-chronic dermal repeated dose toxicity study is not required, as available short term toxicity studies (oral and dermal), as well as the available oral repeated dose toxicity studies together with the low log Pow (<0) indicate low dermal toxicity after repeated application. Additionally, the repeated dose toxicity was adequately addressed by the oral route (see section 7.5.1), allowing evaluation of systemic toxic effects after dermal exposure.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
In accordance with Regulation EC 1907/2006 Annex IX (section 8.6.2), a sub-chronic dermal repeated dose toxicity study is not required, as available short-term dermal toxicity studies, as well as the available skin irritation and sensitisation studies indicate no local toxic effects to the skin.

Justification for classification or non-classification

Based on the results of the oral 90 -day repeated dose toxicity study ( NOAEL = 1000 mg/kg bw/day) the test item is not classified according to EU Directive 67/548/EEC or EU Regulation (EC) No 1272/2008.