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Administrative data

Description of key information

Repeated dose oral. OECD 422. No adverse effects observed up to 1000 mg/kg/day, the highest dose tested. Reliability = 1.
No repeated dose dermal data available. No toxicity in dermal irritation or sensitisation study and dermal route not more acutely toxic than oral.
No repeated dose inhalation data available. Inhalation route not more acutely toxic than oral.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Study included FOB and motor activity endpoints.
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (2000)
Deviations:
yes
Remarks:
Study included FOB and motor activity endpoints.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 65 days
- Weight at study initiation: 300-357 grams (males) and 215-260 grams (females)
- Housing: Solid-bottom cages contained bedding material, and a nestlet and nylabone for enrichment. Sexes were placed on separate racks (except during mating).
- Diet (e.g. ad libitum): ad libitum, except when fasted for clinical pathology
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Approximately 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26ºC
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): approximate 12-hour light/dark cycle
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared by mixing the test substance into the vehicle control substance. Formulations were prepared daily. Formulations were stored at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test Formulation Sampling: Near the beginning of the study, dose formulations were sampled and analyzed for verification of concentration, homogeneity, and stability. Near the middle and end of the study, dose formulations were sampled for an additional concentration verification check.

Method of Analysis: Analysis was performed by 31P NMR Spectroscopy. Samples were shipped on the day they were collected at ambient temperature for NMR analysis. At the time of analysis, the samples were diluted with an appropriate solvent and analyzed by 31P Nuclear Magnetic Resonance Spectroscopy.

Analyses results show that the test substance was homogeneously mixed at the targeted concentrations, and stable in the vehicle under the storage conditions for all dose levels for the study. Test substance was not detected in the control samples.
Duration of treatment / exposure:
All animals were dosed once daily by gavage for approximately 14 days prior to cohabitation and during the cohabitation period (up to 2 weeks). Male and female rats showing no evidence of copulation continued to be dosed after the end of the cohabitation period until sacrifice. Females showing evidence of copulation were dosed throughout gestation. Pregnant females in the process of delivery or showing signs of delivery were not dosed. Females were dosed after delivering litters, until day 3 postpartum. Females that did not deliver a litter continued to be dosed until the day before sacrifice.
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
12 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previous acute oral toxicity study (up and down procedure) in which the test substance was dosed neat, the LD50 value was determined to be >5000 mg/kg in female rats. Based on this data, doses of 100, 300, and 1000 mg/kg/day were selected for the current study. These dose levels include the limit dose level of 1000 mg/kg/day as specified by the relevant testing guidelines. The low and intermediate dose levels were selected to enable detection of potential dose responses across a wide range of dose levels.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily, at least 2 hours post-dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to dosing on test day 0 (baseline) and weekly thereafter at approximately the same time of day (± 2 hours); mortality and moribundity check twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to dosing on test day 0 and weekly during premating and postmating; gestation days (GD) 0, 7, 14, and 20 and lactation days (LD) 0 and 4 and at neurobehavioral evaluations and scheduled sacrifice

FOOD CONSUMPTION:
- Food consumption for each animal determined (g/food/animal/day): Yes; Weekly during premating; GD 0, 7, 14, and 20; and LD 0 and 4. Food consumption was not measured during the postmating period for males, and for females that did not mate.

FOOD EFFICIENCY:
- Calculated as g weight gained/g food consumed: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Test day 14
- Anaesthetic used for blood collection: Yes; Carbon dioxide or Isoflurane
- Animals fasted: Yes
- How many animals: 5/sex/group
- Parameters in table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Test day 14
- Animals fasted: Yes
- How many animals: 5/sex/group
- Parameters in table No. 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes (refer to Section 7.9.1, DI.K1.Neuro.D-18511-1422 for additional details).

OTHER: Coagulation
- Time schedule for collection: at sacrifice
- How many animals: 5/sex/group
- Anaesthetic used: Carbon dioxide

For additional details regarding observations and examinations of reproductive parameters refer to Section 7.8.1 (DI.K1.1Gen.Screen.RD/REPRO/DEV.R.D-18511-1422.KD) and 7.8.2 (DI.K1.DEV.Screen.RD/REPRO/DEV.R.D-18511-1422.KD)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3)
HISTOPATHOLOGY: Yes (see table 3)

Gross examinations were performed on all adult rats. Final body and organ weights were recorded for all rats euthanized at the scheduled sacrifice. Microscopic examination of all reproductive organs was conducted for all rats in the control (0 mg/kg/day) and high-treatment (1000 mg/kg/day) groups and for all reproductive failures (male and female pairs). One mated pair (300 mg/kg/day) required a caesarean section and was also evaluated microscopically. Microscopic examination of all remaining tissues collected was limited to the first 5 surviving rats in the male and female high-treatment and control groups. All collected tissues were also examined microscopically for the 2 decedents. Most gross lesions recorded at necropsy were evaluated microscopically. Gross lesions which were unlikely to have a microscopic correlate (e.g., wet, stain) were not examined.
Statistics:
Statistical analysis was conducted such that data from each group were compared to the vehicle control group using an appropriate pairwise analysis (see Table 4). Male and female data were evaluated separately. The level of significance selected was p <0.05. For each parameter analyzed with a trend test, the test was applied to the data sequentially. If a significant dose-response was detected, data from the top dose group was excluded and the test repeated until no significant trend was detected. For additional details regarding statistics of reproductive and neurotoxicity parameters refer to Section 7.8.1 (DI.K1.1Gen.Screen.RD/REPRO/DEV.R.D-18511-1422.KD) and 7.8.2 (DI.K1.DEV.Screen.RD/REPRO/DEV.R.D-18511-1422.KD), and 7.9.1 (DI.K1.Neuro.D-18511-1422)., respectively
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
One male rat (0 mg/kg/day) was euthanized on day 19 because of eye trauma. One female rat (300 mg/kg/day) was euthanized on day 13 because of hind limb swelling. Both were euthanized for humane reasons. Neither of these deaths was attributed to the test substance.

There were no test substance-related clinical observations in males or females at any dose level tested. In P0 males, there were low incidences of clinical signs that were attributed to injuries incurred during orbital sinus blood collection and included corneal opacity, dark colour, and exophthalmus. All other observations were unremarkable, observed with low frequency, and/or the incidences were not dose dependent.

HAEMATOLOGY
The following statistically significant change in a group mean coagulation parameter was considered to be unrelated to treatment and non-adverse because it did not occur in a dose-related pattern: Minimally prolonged activated partial thromboplastin time (APTT) in female rats dosed with 300 mg/kg/day (120% of control).

CLINICAL CHEMISTRY
The following statistically significant change in a mean clinical chemistry parameter was not adverse or not related to exposure to the test substance: Potassium (K) was minimally decreased in male rats dosed with 1000 mg/kg/day (90% of control). The individual values for potassium in rats dosed with 1000 mg/kg/day, when compared to individual values from other dose groups including controls, were similar. There were no changes in other electrolyte measurements (sodium, chloride) nor were there decreases in female rats at this same dose. Therefore, this minimal decrease in potassium was considered to be unrelated to treatment and thus non-adverse.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at the highest dose tested.
Critical effects observed:
not specified
Conclusions:
NOAEL = 1000 mg/kg/day

Executive summary:

The objective of this study was to evaluate the potential subchronic and reproductive/developmental toxicity of the test substance when administered by oral gavage to male and female rats during premating, cohabitation, gestation, until postnatal day (PND) 3. Four groups of parental (P0) male and female Crl:CD(SD) rats (12/sex/group) were administered formulations of the test substance via once daily oral gavage for 14 consecutive days prior to mating, throughout mating, and then continuing through one day prior to the day of euthanasia. The dose levels administered were 0, 100, 300, or 1000 mg/kg/day. Samples of the dose formulations were analyzed and confirmed that the formulations were at targeted concentrations, homogeneous, and stable under the conditions of use.  During the in-life phase of the study, all animals were observed twice daily for appearance and behaviour. Clinical observations, body weights, and food consumption were recorded at appropriate intervals for Pmales throughout the study and for Pfemales prior to mating and during gestation and lactation. Neurobehavioral examinations consisting of an abbreviated functional observational battery (FOB) and motor activity (MA) were performed for all P0 animals once prior to dosing to provide baseline data and once more near the end of the premating period. In addition, clinical pathology data consisting of haematology, coagulation, and clinical chemistry data were collected prior to the end of the premating period. All Pfemales were allowed to deliver and rear their pups until weaning on PND 4. Each Pparental animal received a complete detailed gross necropsy and selected organs were weighed and/or retained for histopathologic examination.

 

There was no evidence of test substance-related toxicity at any dose level tested. The in-life data, including body weight and food consumption parameters, clinical observations, neurobehavioural endpoints (FOB and MA), clinical pathology, as well as reproductive performance data including mating and fertility indices, gestation length, offspring viability, litter sizes, litter sex ratio, and pup weights, were comparable across all groups throughout the study. In addition, there were no test substance-related effects on either gross or microscopic histopathology or on organ weights. 

 

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for general repeated-dose toxicity was 1000 mg/kg/day. This conclusion is based on the lack of any evidence of any adverse and/or test substance-related effects at any dose level tested.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat

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

Additional information

No evidence of systemic toxicity was observed following repeated dose exposure by oral gavage. The NOAEL was 1000 mg/kg/day.

Justification for classification or non-classification

Based on the lack of adverse effects in the repeat dose study and in the acute oral, dermal, and inhalation exposures, the substance does not need to be classified for repeated dose toxicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.