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Description of key information

Oral (OECD 422), rat: NOAEL = 100 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 May - 21 Sep 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD), SPF
Details on species / strain selection:
Sprague-Dawley rats are commonly used in both the general systemic toxicity and reproductive and developmental toxicity studies with a large historical control data base. In addition, the rat is a required species in the regulatory guidelines.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 9 weeks (male), 8 weeks (female)
- Weight at study initiation: 312.7-358.4 g (males), 189.3-228.5 g (females)
- Housing: Acclimation period and pre-mating: 1 animal per cage; Mating: 1:1; Lactation: neonates were kept with the dam; animals were kept in stainless wire mesh cages (260W x 350D x 210H mm) and in polycarbonate cages (260W x 420L x 180H mm)
- Diet: Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C (Harlan Laboratories, Inc., USA), ad libitum
- Water: public tap water filtered and irradiated by ultraviolet light, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 - 23.8
- Humidity (%): 46.3 - 65.6
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed with a small amount of vehicle to dissolve using a magnetic stirrer and then, the vehicle was gradually added to yield the desired concentration. The dosing formulations were stored in a refrigerator (4.4–5.5 °C). These dosing formulations were used within 7 days.

VEHICLE
- Justification for use and choice of vehicle: Through the preliminary solubility test to determine the solubility and dispersion characteristics of the test substance, corn oil was selected as the vehicle because the test substance was dissolved in it.
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the dosing formulations were conducted using gas chromatography and samples were taken three times from the middle of each dosing formulation prior to dosing and analyzed for verification of dose level concentration. The results of dose concentration analyses were determined to be 97.13 – 99.87%. These results were within the acceptable limits (± 15% of nominal values). As a result of homogeneity and stability analyses conducted the 0.2 and 200 mg/mL dosing solutions were confirmed to be homogenous and stable for 4 h at room temperature and for 7 days under refrigeration.
Duration of treatment / exposure:
Main groups:
males: for 6 weeks, starting 2 weeks before mating, during mating and 2 weeks after mating
females: for 2 weeks prior to mating, throughout gestation and for at least 4 days after delivery up to the day before the scheduled terminal necropsy

Recovery groups:
Males and females of recovery groups were dosed once daily for 6 weeks. Animals were not mated and were assigned to 2 weeks of recovery period after the completion of administration.
Frequency of treatment:
once daily, 7 days/week
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 (main groups)
6 (recovery groups; for control and high dose groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previously conducted 2-week repeated oral dose range-finding study, two males and five females were found moribund or dead at 600 mg/kg bw/day on Day 2. There were no test substance-related effects in animals at 200 mg/kg bw/day. Therefore, the high dose level was selected at 300 mg/kg bw/day. Then, the mid and low dose levels were selected at 100 and 30 mg/kg bw/day, respectively.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily mortality/viability
- Cage side observations included: All animals were observed for general condition and clinical signs at least once daily throughout the study. Females were also observed for signs of abortion and pre-mature birth.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations for signs and symptoms of adverse effects, including central and autonomic nervous system effects, motor activity and behavior, were conducted on all animals once before the test and once a week throughout the dosing and recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of males of the main group and animals of each sex of the recovery group were recorded just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing and recovery periods, the day before necropsy and on the day of necropsy (fasted body weights). Body weights of females of the main group were recorded just prior to dosing on Day 1, once a week throughout the dosing and recovery periods, on Days 0, 7, 14 and 20 of gestation, on Days 0 and 4 post partum and on the day of necropsy (fasted body weights). Fasted body weights recorded on the day of necropsy were presented, but were not included in statistical analysis.

FOOD CONSUMPTION: Yes
- Food consumptions of males of the main group and animals of each sex of the recovery group were recorded just prior to dosing on Day 1, once a week during the dosing and recovery periods and the day before necropsy. Food consumptions of females of the main group were recorded just prior to dosing on Day 0, once a week throughout the dosing and recovery periods, on Days 0, 6, 13 and 19 of gestation, on Days 0 and 3 post partum. Food consumption was not recorded during mating.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 6 males and females were randomly selected from the main study groups in addition to all animals from the recovery groups.
- Parameters examined: erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), leukocyte count (WBC), neutrophils (NEU), lymphocytes (LYM), monocytes (MONO), eosinophils (EOS), basophils (BASO), reticulocytes (Reti), prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 6 males and females were randomly selected from the main study groups in addition to all animals from the recovery groups.
- Parameters examined: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (Crea), total bilirubin (T-Bili), total protein (TP), albumin (Alb), globulin (Glo), A/G ratio, glucose (Glu), total cholesterol (T-Chol), triglyceride (TG), sodium (Na), potassium (K), chloride (Cl), calcium (Ca)

URINALYSIS: Yes
- Time schedule for collection of urine: 6 males and females were randomly selected from the main groups in addition to all recovery animals for urinalysis two days before necropsy. Fresh, 3-hour and 24-hour urine samples were collected from the selected animals and analyzed.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Animals were fasted during the fresh urine collection, but were allowed free access to drinking water.
- Parameters examined: in fresh urine samples: pH, protein, glucose, bilirubin, occult blood, color and turbidity, sediment; in 24-hour urine samples: urine volume, specific gravity

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected animals were examined a few days before necropsy.
- Dose groups that were examined: 6 males and females were randomly selected from the main study groups in addition to all recovery animals
- Battery of functions tested: pinna reflex, auditory (sound) reflex, corneal reflex, pupillary reflex, grip strength test, rotarod test, spontaneous motor activity test
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All males of the main group were sacrificed 2 weeks after mating and females of the main group were sacrificed on Day 6 post-partum. All animals of the recovery group were sacrificed 2 weeks after final dosing. Non-pregnant females were sacrificed on Day 27 after the last day of mating. Complete gross post-mortem examinations were conducted on all animals including the external and internal surfaces. All grossly visible abnormalities were recorded.

ORGAN WEIGHTS: Yes
Paired organs were weighed together. Animals were fasted overnight prior to necropsy and body weights were recorded on the day of necropsy. Organs were weighed and organ-to-body weight ratios were calculated. The testes and epididymides of all adult males were weighed. 6 males and 6 females were randomly selected from the main study animals in addition to all recovery animals for necropsy. Following organs were weighed: brain, heart, liver, thymus, spleen, kidneys, adrenals, ovaries, uterus

HISTOPATHOLOGY: Yes
Tissue preservation and slide preservation
6 males and 6 fmales were randomly selected from the main groups in addition to all recovery animals for tissue preparation. The testes and epididymides were fixed in Bouin's solution. The eyes with optic nerves were fixed in Davidson’s fixative. All other tissues were preserved in 10% neutral buffered formalin.

For the histopathological examination, the preparation of specimens of organs and tissues was carried out and the remaining organs and tissues preserved in 10% neutral buffered formalin: brain, pituitary, thymus, lung with bronchi, trachea, thyroid, esophagus, heart, liver, spleen, kidneys, adrenals, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, testes, epididymides, prostate, ovaries, uterus, submandibular lymph node, mesenteric lymph node, bone marrow (femur and sternum), spinal cord, sciatic nerve, eye and optic nerve, urinary bladder

Besides, from all animals except for six females and males in the main group, the following organs and tissues were harvested and preserved: brain, pituitary, heart, thymus, liver, spleen, kidneys, adrenals, prostate, testes, epididymides, ovaries, uterus

Histopathological examinations were conducted as follows:
- 6 males and females from the control, low, mid and high dose group (especially, focused on spermatogenesis and interstitial testicular cell structure)
- All tissues from animals found dead or killed in a moribund condition
- All gross, macroscopic lesions
- Target organs noted at the high dose were examined for the recovery group (liver)
Statistics:
The statistical analysis of this study was conducted using the SAS program (SAS 9.3). For the data including body weights, food consumption, urine volume and specific gravity, hematology and blood biochemistry parameters, organ weights, mating result, birth and survival rates, sensory reactivity and motor activity, the Bartlett test was conducted to test for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) test was employed on homogeneity, if significant (significance level: 0.05), followed by Dunnett’s t-test for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test was employed on heterogeneity, if significant (significance level: 0.05), followed by Steel’s test for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Mating index, fertility index and other data associated with gestation were analyzed utilizing Fisher’s exact test (significance levels: 0.05 and 0.01). For the data of the recovery group, Folded-F test was employed to test homogeneity of variance (significance level: 0.05, two-tailed). Student t-test was employed on homogeneity, if overruled, Aspin-Welch t-test was applied (significance levels: 0.05 and 0.01, two-tailed).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
temporary loss of locomotor activity was observed in females at 300 mg/kg bw/day
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Clinical biochemistry findings:
effects observed, non-treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increase of liver weights at 300 mg/kg bw/day in both sexes
Gross pathological findings:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
reversible hepatocellular hypertrophy in the centrilobular zone of males at 100 mg/kg bw/day and in both sexes at 300 mg/kg bw/day
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
In the main and recovery groups, all animals survived the duration of the study. There was no effect on the mortality.
In the main group, loss of fur was observed in one male at 30 mg/kg bw/day on Days 41 and 42. A decrease in locomotor activity, dirty nose and/or soiled perineal region were observed in one female after parturition (from Day 1 to 3 post partum). Wound (right shoulder) was observed in one male at 100 mg/kg bw/day from Day 40 to Day 42. Temporary loss of locomotor activity was observed frequently in females at 300 mg/kg bw/day from GD 7 to the end of dosing. In the recovery group, temporary loss of locomotor activity was observed frequently in females at 300 mg/kg bw/day from Day 25 to Day 37. Salivation was shown in two males at 300 mg/kg bw/day from Day 39 to Day 42.
In the recovery group, temporary loss of locomotor activity was observed frequently in females at 300 mg/kg/day from Day 25 to Day 37. Salivation was shown in one male at 300 mg/kg bw/day on Days 39 and 42. During the recovery period, abnormal clinical signs were not observed in both sexes at 300 mg/kg bw/day.
The symptoms such as wound and salivation were considered to occur due to individual difference because they were observed temporarily.
Temporary loss of locomotor activity observed in females at 300 mg/kg/day in the main and recovery groups was considered to be a test substance-related effect.

No clinical abnormalities in detailed examinations were observed in males and females of the main and recovery groups.

BODY WEIGHT AND WEIGHT GAIN
No statistically significant differences in body weight changes were noted in males and females of the main and recovery groups compared to the control group.

FOOD CONSUMPTION
A statistically significantly increase in food consumption was noted in males at 300 mg/kg bw/day in the main group on Day 14. A statistically significantly increase in food consumption was noted in females at 300 mg/kg bw/day in the recovery group on Day 49. These statistical significances were not considered to be test substance-related changes since these were differences of small magnitude and they were not related to body weight changes.

HAEMATOLOGY
In the main and recovery groups, no effects were noted in any animal in any dosing group.
Other statistical significances were not considered to be test substance-related changes since these were differences of small magnitude and they were within the range of historical reference data.

CLINICAL CHEMISTRY
In the main and recovery groups, no effects were noted in any animal in any dosing group.
Other statistical significances were not considered to be test substance-related changes since these were differences of small magnitude and/or they were within the range of historical reference data.

URINALYSIS
In the main and recovery groups, no effects were noted in any dosing group.

NEUROBEHAVIOUR
In the main and recovery groups, there were no test substance-related effects on auditory reflex, pinna reflex, pupillary reflex and corneal reflex test in both sexes in the test substance dosing groups when compared to the control group.
In the main and recovery groups, there were no test substance-related effects on rotarod test and spontaneous motor activity in both sexes in the test substance dosing groups when compared to the control group.
In the main group, statistically significantly decreased forelimb grip strength was noted in males at 30 mg/kg bw/day when compared to control group. However, it was not considered to have meaning of toxicological significance since it was difference of small magnitude and without dose-dependency.

ORGAN WEIGHTS
In the main group, increases in the absolute (+36%) and relative (+25%) organ weights of the liver were noted in males at 300 mg/kg bw/day. Relative liver weights were also increased in females (+25%) at 300 mg/kg bw/day. While the increase in the liver organ weight was not considered to be a test substance-related adverse effect since it was not accompanied by increases of ALT, AST and ALP and it was reversible in organ weights in the recovery group, the absolute liver weight increase was considered to exceed a level that should be cosidered adaptive.
Other statistical significances in the absolute and/or relative organ weights were not considered to be test substance-related effects since the differences were small in magnitude and they were within the historical range limit.

GROSS PATHOLOGY
Macroscopic examination at necropsy did not reveal any treatment-related changes. All other macroscopic findings observed in this study were considered to be incidental, and not related to the test substance.

HISTOPATHOLOGY
Following the dosing period, test substance-related microscopic findings were present in the liver. Hepatocellular hypertrophy was observed in males at 100 mg/kg bw/day and in both sexes at 300 mg/kg bw/day after six weeks of treatment. The hepatocellular hypertrophy was characterized by increased cytoplasmic volume, which was within centrilobular zone. At the end of the 2-week recovery period, this finding disappeared at 100 and 300 mg/kg bw/day. The hepatocellular hypertrophy was not considered to be adverse, since the hepatocellular hypertrophy in centrilobular zone is generally considered to be an adaptive response. Furthermore, the effect was reversible within the recovery period of the study.
All other microscopic findings seen in various organs and tissues were considered to be incidental and of no toxicological significance.
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at 100 mg/kg bw/day
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1. Absolute and relative liver weights (n=6)

 

Dose level (mg/kg bw/day)

0

30

100

300

Male

Female

Male

Female

Male

Female

Male

Female

Liver weight

 

Absolute (g)

12.93 ± 1.64

11.13 ± 1.67

13.07 ± 1.67

11.58 ± 1.11

14.56 ± 2.05

12.69 ± 1.18

17.61 ± 1.68**

12.59 ± 0.84

Relative (%)

2.75 ± 0.20

3.79 ± 0.30

2.75 ± 0.22

3.85 ± 0.26

3.01 ± 0.13

4.11 ± 0.20

3.43 ± 0.17**

4.72 ± 0.30**

Significantly different from control group by Dunnett's test: *p<0.05, **p<0.01

 

Table 2. Incidence of treatment-related microscopic findings

 

Dose level (mg/kg bw/day)

0

30

100

300

Male

Female

Male

Female

Male

Female

Male

Female

Liver

 

Hypertrophy, hepatocellular, centrilobular

0/6

0/6

0/6

0/6

1/6

(minimal)

0/6

5/6

(minimal)

5/6

(1x mild,

4x minimal)

Conclusions:
Based on the results of this study, the NOAEL for systemic toxicity was set at 100 mg/kg bw/day for males and females due to effects on liver weight.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.
System:
hepatobiliary
Organ:
liver

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

The test substance was tested in a combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD Guideline 422 and in compliance with GLP (2015). Twelve Sprague Dawley rats per sex and dose were treated via gavage with test substance at concentrations of 30, 100 and 300 mg/kg bw/day, respectively. The control group received the vehicle corn oil. Additionally, a recovery group of 6 rats per sex was allocated to the control and high dose group. Males were treated for 6 weeks, starting 2 weeks before the mating period, during mating and 2 weeks after mating. Females were treated for 2 weeks prior to mating, throughout gestation and for at least 4 days after delivery up to the day before the scheduled terminal necropsy. The doses were selected on the basis of a 2-week repeated oral dose range-finding toxicity study in which two males and five females were found moribund or dead at 600 mg/kg bw/day on Day 2. No test substance-related effects were observed in animals at 200 mg/kg bw/day.

In the main study, all animals of the main group and all animals of recovery group survived the duration of the study.Temporary loss of locomotor activity was observed in females at 300 mg/kg bw/day for the main and recovery group. No test substance-related adverse effects were noted in the results of detailed clinical signs, body weights, food consumption, sensory function, motor activity, urinalysis, hematology, blood chemistry, and necropsy in parent animals.

In the main group, increases in the absolute (+36%) and relative (+25%) organ weights of the liver were noted in males at 300 mg/kg bw/day. Relative increase in liver weight (+25%) was also observed in females at 300 mg/kg bw/day. No significant findings were seen for the organ weight in the male recovery group. Hepatocellular hypertrophy was observed in males at 100 mg/kg bw/day and in both sexes at 300 mg/kg bw/day in the main group. While this finding was not considered to be adverse since the hepatocellular hypertrophy in centrilobular zone is generally considered to be an adaptive response, and had completely reverted after recovery period, the absolute liver weight increase was considered to exceed a level that should be considered adaptive.

Based on these results, the NOAEL for the test substance for systemic toxicity was considered to be 100 mg/kg bw/day due to the absolute liver weight increase and the temporary loss in locomotor activity observed at 300 mg/kg bw/day.

Justification for classification or non-classification

The available data on repeated oral dose toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification. The increase in liver organ weights was reversible in the recovery group and not accompanied by changes in clinical parameters or organ dysfunction. Despite the temporary loss in locomotor activity, no test substance-related adverse effects were noted in the results of detailed clinical signs, in sensory function and motor activity. Thus no STOT RE classification was considered.