Registration Dossier

Administrative data

Description of key information

Based on results of an OECD 422 and GLP compliant study, the toxicological NOAEL for daily oral gavage of the test item in male and female rats for 28 consecutive days was found to be 300 mg/kg bw/d. Dose levels of 100, 300 or 1000 mg/kg bw/day were applied. Effects of general toxicity were observed at the High dose only and were completely reversible within the recovery period.

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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-02-16 to 2016-11-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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:
Sprague-Dawley
Details on species / strain selection:
The rat (Rattus norvegicus) was selected to satisfy the regulatory requirements for toxicity testing in a rodent species. The current state of scientific knowledge and the applicable guidelines do not provide acceptable alternatives, in vitro or otherwise, to the use of live animals to accomplish the purpose of this study. The development of knowledge necessary for the improvement of the health and well-being of humans as well as other animals required in vivo experimentation with a wide variety of animal species. Whole animals are essential in research and testing because they best reflect the dynamic interactions between the various cells, tissues, and organs comprising the human body. The rat is an universally used rodent model for evaluating the toxicity of various classes of chemicals and for which there is a large historical database.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Canada Inc., St-Constant, Quebec, Canada
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 10 wks
- Weight at study initiation: (P) Males: 411 - 506 g; Females: 256 - 322 g;
- Housing: individually, except during mating
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least two weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 50 +/- 20 %
- Air changes: 10 - 15 per hr
- Photoperiod: 12 /12 hrs dark / hrs light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item and vehicle dosing formulations were prepared at least weekly. The test item was weighed and transferred into a container. The vehicle was added (Q.S.), followed by stirring.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stir plate.

VEHICLE
- Justification for use and choice of vehicle: corn oil is a vehicle recommended by guideline and provides good solubility of the test item.
- Concentration in vehicle: 0, 25, 75, 250 mg/mL
- Amount of vehicle: 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of Dosing Formulations:
Stability of the dosing formulations under the conditions of use during the study and covering the range of concentrations and storage used in the study was assessed. Preliminary results confirmed that test item formulations in corn oil at concentrations between 25 and 250 mg/mL are stable at room temperature and when stored frozen at approximately – 20°C.

Analysis of Test Item Concentration:
To verify the concentration and homogeneity of the test item in the formulations, representative samples (5 mL in duplicate) were taken from the top, middle, and bottom of each concentration at the start of the study, and from the middle of a preparation conducted towards the end of the treatment period. The samples were stored frozen (set maintain to -20°C). The analysis was performed by gas chromatography. Acceptance criteria for the formulation were ±15% of nominal.
Duration of treatment / exposure:
Males: 14 days prior mating; up to 14 days during mating; followed by 14 day recovery period
Females: 14 days prior mating; up to 14 days during mating; during gestation (GD 0 through to Day 4 post partum); followed by 14 day recovery period
Frequency of treatment:
daily
Dose / conc.:
0 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)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dosages were selected based on preliminary dose range finding study
- Rationale for animal assignment: random
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Males and Females: on day of randomization and weekly through treatment and recovery period; Females additionally on GD 0, 7, 10, 14, 17, 20 and Day 4 post partum; on day of scheduled necropsy a fasted body was recorded.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: end of dosing period
- Dose groups that were examined: 5 animals/sex of control and high dose

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Yes: Isoflurane
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters examined: Hematocrit; Platelet count; Hemoglobin Plateletcrit / thrombocrit; Hemoglobin distribution width; Red blood cell count; Mean corpuscular hemoglobin; Red cell distribution width; Mean corpuscular volume; Reticulocyte counts (absolute and relative); Mean corpuscular hemoglobin; Concentration; WBC differential (absolute and relative); White blood cell count (WBC)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters examined: A/G ratio (calculated); Creatinine; Alanine aminotransferase; Globulin (calculated); Albumin; Glucose; Alkaline phosphatase; Phosphorus (inorganic); Aspartate aminotransferase; Potassium; Bilirubin (total) Sodium; Total Calcium; Total protein; Chloride Triglycerides; Cholesterol (total); Bile Acids; Urea

URINALYSIS: Yes
- Time schedule for collection of urine: prior to necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Bilirubin; pH; Blood Protein; Color and appearance; Specific gravity; Glucose; Urobilinogen; Ketones Volume; Microscopic analysis

NEUROBEHAVIOURAL EXAMINATION: Yes: FOB
- Time schedule for examinations: last week of dosing and recovery period
- Dose groups that were examined: control and high dose group
- Battery of functions tested: open-fiel evaluation / manipulative evaluation / physiological evaluation / neuromuscular evaluation / motor activity

IMMUNOLOGY: No

OTHER:
-Coagulation: The following parameters were measured on blood samples collected into tubes containing citrate as anticoagulant. Targeted volume of blood collected for each sample was 1.0 mL: Activated partial thromboplastin time; Prothrombin time
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Other examinations:
- Estrous cyclicity: At least 7 days prior to the start of treatment and during mating until the day of positive mating was determined.

- Sperm parameters: Parameters examined in P male parental generations: testis weight, epididymis weight, seminal vesicles with coagulating glands, stages of spermatogenesis, sperm morphology, retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen. Examination of the intact epididymis included the caput, corpus, and cauda. The epididymis was evaluated for leukocyte infiltration, change in prevalence of cell types, aberrant cell types, and phagocytosis of sperm.

LITTER OBSERVATIOS:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Statistics:
please refer to "Any other information on materials and methods"
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The incidence of salivations (slight to moderate) was observed with 15 of 17 females and 17 of 17 males dosed with the test item observed at 1000 mg/kg bw/day. Other clinical signs observed were transient, isolated or associated with animals found dead or euthanized pre-terminally or observations that are commonly identified in the species.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One animal was found dead in the Control group on Day 24. One animal of the Low (100 mg/kg bw/day) and High Dose group (1000 mg/kg bw/day) were euthanized due to poor and deteriorating conditions on GD 24 and GD 23, respectively. Test item-related microscopic changes in the Group 4 female included marked tubular degeneration/necrosis with concurrent mild to moderate tubular cast formation and tubular dilatation in the kidney and minimal hepatocellular hypertrophy in the liver. Test item-related microscopic change in the Group 2 female included minimal tubular vacuolation in the kidney, minimal hepatocellular hypertrophy in the liver, mild multifocal mucosa erosion/ulceration in the glandular stomach, and mild crypt epithelium hyperplasia/hypertrophy in the cecum mucosa.
Although the microscopic changes described above may have contributed in part to the poor clinical conditions of the euthanized low and high dose female animals, the prolongation of the gestation period (failure to litter) and microscopic findings associated with stress (cited below), were considered the primary cause of moribundity. Hence, euthanasia of these animals was considered secondary to complications from physiologic stress and not directly related to the test item. Other microscopic changes in both rats were considered to be, predominantly, stress-related and/or secondary, and not directly test item-related. Physiological stress-related minimal adrenal cortical hypertrophy was noted in both rats with secondary minimal to moderate lymphoid hypocellularity and/or lymphoid degeneration/necrosis in lymphoid tissue (thymus, spleen, lymph nodes, Gut Associated Lymphoid Tissue (GALT)) and minimal bone marrow hematopoietic hypocellularity. Microscopic findings frequently correlated with macroscopic findings in these two females.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights trended lower for males dosed with test item from Day 7 of the dosing period and attained statistical significance (p ≤ 0.05 or p ≤ 0.01) at 1000 mg/kg bw/day from Day 21 to 49 (including recovery period) in comparison to the controls. The reduction in body weights for these animals resulted in a statistical (p ≤ 0.05, p ≤ 0.01 or p ≤ 0.001) significant reduction of weekly body weight gains from Day 0 to 28 in comparison to the controls. There were no statistical adverse effects on body weight or body weight changes for females during the premating, mating, gestation or post-partum periods up to 1000 mg/kg bw/day in comparison to the Controls.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There was no adverse effect on food intake for males up to Day 49 or for females during the gestation or post-partum periods dosed with test item up to 1000 mg/kg bw/day.
There were statistically significant (p ≤ 0.05) increase food consumption for females dosed with test item at dose levels of 300 mg/kg bw/day from gestation days 7 to 14 and a statistically significant increase of food consumption from gestation days 7 to 20 (p ≤ 0.05 or p ≤ 0.01) for females dosed at 1000 mg/kg bw/day, in comparison to the controls. There was also a statistical significant (p ≤ 0.05) decrease of food consumption during the post-partum period and a statistically significant increase during days 28 to 35 for females dosed at 1000 mg/kg bw/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item related changes included slight reductions in mean platelet values (and plateletcrit) at the high dose (1000 mg/kg bw/day) in males on Day 29 (statistically-significant) and in females (Day 5 post-partum) compared to control means. Additional findings included reduced mean eosinophil counts (and percent of leukogram) in males on Day 29 (statistically significant) compared to the control mean. Both changes were not observed in recovery animals. Other minor statistically-significant changes were noted at main and at recovery periods that were not considered related to treatment with the test item due to an absence of a doseresponse, and/or minimal changes considered within biological variation.

Test item related coagulation parameter changes included an observed statistical significant increase (p ≤ 0.001) in mean Prothrombin Time (PT) for males dosed at 1000 mg/kg bw/day at the end of the dosing period (Day 29) in comparison to the Controls; this was considered slight and was not observed at the end of the recovery period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There was a statistical significant increase (p ≤ 0.05 or p ≤ 0.01) of mean serum total bile acids and alkaline phosphatase activity at the end of the dosing period (Day 29) for males at 1000 mg/kg bw/day in comparison to the control means. There were also increased serum triglycerides in individual high dose males. These changes were considered test item-related and of no apparent relationship to the minimal histologic findings of mononuclear cell infiltrate with or without minimal single hepatocellular necrosis in the liver of high dose males.
Other statistical changes noted in clinical chemistry parameters were not considered related to the test item and included a reduction of mean serum urea (p ≤ 0.05) for high dose males and females at the end of the recovery period in comparison to the controls, and a statistical significant increase (p ≤ 0.05) in mean serum sodium at the end of the dosing period (Day 29) for males at 300 mg/kg bw/day in comparison to the controls.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no adverse effects of test item on urinalysis parameters.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no adverse effects on qualitative or quantitative functional observation battery at any dose level up to 1000 mg/kg bw/day. A statistical significant reduction (p ≤ 0.05) of fore limb grip strength was observed for females at 1000 mg/kg/day during the last week of dosing and for both fore and hind limbs of males (p≤ 0.05 or p≤ 0.01) at the end of the recovery period in comparison to the controls. The reduction cited above and any other observed statistically significant changes were considered incidental or of no toxicological importance.

There were no test item-related effects on the motor activity test at any dose level up to 1000 mg/kg bw/day for males or females.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Intergroup organ weight change clearly related to administration of the test item was noted in the liver (females) and thymus (males) of Main rats. Statistically and/or non-statistically significant liver weight change was noted primarily in female rats. Group mean liver weights (absolute and relative to brain and body weight) were higher in female rats dosed at 1000 mg/kg/day in comparison compared to the group mean weights of the female control.

The slight change in mean liver weights noted in males was without a dose-response and where significantly increased (relative to body weight) may have been affected by the lower mean body weight compared to the control group. Microscopic findings however were noted in the liver of male rats. There was no microscopic change that correlated with the higher liver weight in female rats. Statistically significant thymus weight change was noted in Main male rats. Group mean thymus weights (absolute and relative to brain and body weight) were lower in male rats dosed at 1000 mg/kg/day in comparison to the group mean weights of Controls. Microscopic findings in the thymus of male rats correlated with the lower thymus weights.

Similar changes were not observed in females. Thymus weights in high dose recovery animals were comparable to controls; however, liver weights were significantly reduced in high dose males at the end of the recovery period.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There was no macroscopic change related to administration of test item in Main or Recovery animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic changes in Main male rats were observed in the testes, liver, and thymus of male rats dosed at 1000 mg/kg bw/day. Microscopic changes in Main female rats were observed in the brain, kidney, cecum, and thymus of one female rat dosed at 1000 mg/kg bw/day. Minimal retained spermatids (Stage 9, 10) were noted in the testis of 5/12 Main male rats dosed at 1000 mg/kg/day. Minimal mononuclear infiltrate with or without concurrent minimal hepatocellular single cell necrosis was noted in the liver of 3/5 Main male rats dosed at 1000 mg/kg bw/day. Minimal lymphoid hypocellularity was noted in the thymus of 3/5 Main male rats dosed at 1000 mg/kg bw/day and were considered test item-related, correlating with lower group mean thymus weights.
Microscopic changes in Main female rats were observed in one female rat dosed at 1000 mg/kg bw/day of that included: brain, minimal to mild poliomalacia, neuronal degeneration, and gliosis involving the hippocampus; kidney, mild tubular hyaline droplets; cecum, minimal mucosa crypt epithelium hyperplasia/hypertrophy, and; thymus, marked lymphoid hypocellularity. The characteristics of the hyaline droplets noted in the kidney of this female rat were considered to be different than spontaneous hyaline droplets noted in recovery control male rats and, therefore, test item-related. Minimal lymphoid hypocellularity was noted in the thymus of females from all treatment groups, including control Group 1 Main female rats, and was, therefore, considered to be a spontaneous background change and, therefore, distinct from the test item-related marked lymphoid hypocellularity observed in this one Group 4 female rat.
Minimal retained spermatids (Stage 9) were noted in the testis of 1/5 Recovery male rat dosed at 1000 mg/kg bw/day. The lower incidence in Recovery males, compared to Main males, indicated incomplete but progressive ongoing reversal following a recovery period. Microscopic changes noted previously in the liver, kidney, cecum, thymus, and brain of Main male and/or female rats dosed at 1000 mg/kg bw/day reversed completely during the recovery period.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
- Estrous cycle: no effects observed

- Sperm measures: Microscopic changes in Main male rats were observed in the testes. Minimal retained spermatids (Stage 9, 10) were noted in the testis of 5/12 Main male rats dosed at 1000 mg/kg bw/day.
Minimal retained spermatids (Stage 9) were noted in the testis of 1/5 Recovery male rat dosed at 1000 mg/kg bw/day. The lower incidence in Recovery males, compared to Main males, indicated incomplete but progressive ongoing reversal following a recovery period.

- Reproductive performance:
The mating indices were 100 % for all males and females in all groups including the controls. The fertility indices were 83.3, 91.7, 100 and 50% for control, 100, 300 and 1000 mg/kg bw/day, respectively for males and females.
The gestation indices were 100, 90.9, 100 and 66. 7% for the Control, 100, 300 and 1000 mg/kg bw/day, respectively and post-implantation loss was 4-fold higher for females at 1000 mg/kg bw/day in comparison to the Controls.
There was a statistical significant (p ≤ 0.05) increase mortality index and number of dead pups at 1000 mg/kg bw/day in comparison to the controls.
The mean gestation length was prolonged and was statistically significant (p ≤0.001) for females dosed with test item at 1000 mg/kg bw/day (23.3 days) in comparison to the control that were 21.8 days.
There were no other adverse significant effects on maternal performances including total corpora lutea, implantation sites, sex ratio, live birth and malformed pups.
The number of pups at birth and number alive on PPD 4 were markedly lower for dams at 1000 mg/kg/day in comparison to the controls. The numbers of male and female pups on PPD 0 or 4 were similar.
The survival index (PPD4) was statistically significantly (p≤ 0.01) reduced for pups at 1000 mg/kg bw/day in comparison to the controls.
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
clinical signs
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
other: reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
clinical signs
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
other: reporductive performance
Key result
Critical effects observed:
no
Conclusions:
The daily administration of the test item by oral gavage to male and female Sprague-Dawley rats at dose levels of 100, 300 or 1000 mg/kg bw/day from 2 weeks prior to mating and during the mating period for up to 14 days through to post-partum day (PPD) 4 in pregnant females, resulted in toxicological findings at the high dose only and included minor reductions in platelets and eosinophils, increases in serum alkaline phosphatase activity and total bile acids, and slightly increased PT. Increased liver weights and reduced thymus weights with microscopic changes in the liver, thymus, and testes of males, and brain, kidney, cecum, and thymus of a single female were noted at the high dose. Microscopic changes reversed completely with the exception of incomplete reversal of retained spermatids in testes. There were adverse effects on male and female fertility, gestation length and post implantation loss and number of live fetuses and pup body weights to PPD 4 at 1000 mg/kg bw/day.
Based on the results of this study, the toxicologic No Observable Adverse Effect Level (NOAEL) for daily oral gavage of the test item in male and female Sprague-Dawley rats was 300 mg/kg bw/day, while the NOAEL for reproductive performance and development of the F1 offspring was also 300 mg/kg bw/day.
Executive summary:

The objective of this study was to obtain information on the toxicity of the test item following repeated (daily) administration by oral gavage to male and female Sprague-Dawley rats at 3 dose levels for:

1) a 14-day period prior to mating,
2) up to 14-days during mating followed by a 2-week recovery period and
3) during gestation in pregnant females from Gestation Day (GD) 0 (representing successful mating) through to Day 4 post-partum.

Additional objectives included determination of possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Post-Partum Day (PPD) 4.

The test item and reference item/vehicle were administered daily, during the dosing period, as shown below:

 

Table 1 Study design

Treatment group

Dose Level (mg/kg bw/d)

Dose Conc. (mg/mL)

Dose Volume (mL/kg)

Number of animals

Main

Recovery**

M

F

M

F

1. Control*

0

0

4

12

12

5

5

2. Test item Low

100

25

12

12

-

-

3. Test item Mid

300

75

12

12

-

-

4. Test item High

1000

250

12

12

5

5

  * Group 1 received the vehicle, corn oil, alone.

** Recovery phase animals not placed for mating.

M = male; F = female

The test item was formulated in corn oil (also the reference item/vehicle). Males and female Sprague-Dawley rats were 10 weeks old and body weights ranged from 411 to 506 g for males and from 256 to 322 g for females at the start of dosing. Males were dosed for a total of 28 days (inclusive of pre-mating and mating periods), females were dosed during the 14-day premating period, during the mating period for up to 14-days and from GD 0 through to Day 4 postpartum. Main study males were euthanized on Day 29. Main study females were euthanized on PPD 5 and Recovery male and female rats were euthanized on Day 56.

Cage side clinical observations were performed twice daily and a detailed clinical observation was performed on days of body weight recordings. Individual body weight was measured on the day of randomization and weekly through the treatment and recovery period for males. Body weights were performed at randomization, once weekly during the pretreatment and treatment period prior to confirmed mating on GD 0, 7, 10, 14, 17 and 20 and post-partum day (PPD 0 and 4) for females. A Functional Observation Battery (FOB), was performed on 5 animals/sex/group prior to their scheduled necropsy during the last week of dosing (during lactation for females), and during the 14-day recovery period. Clinical pathology (hematology, clinical chemistry, coagulation, and urinalysis) was performed on 5 animals/sex/group prior to their scheduled necropsy. Ophthalmoscopy funduscopic and biomicroscopic (slit lamp) examinations were performed on 5 animals/sex from Groups 1 and 4 towards the end of the dosing period. Estrous cycles were determined prior to and during mating. Dams were observed for parturition at least 3 times a day starting from GD 20, litter size and external sex were determined on PPD 0 and 4 and their offspring (F1) had clinical observations, mortality, body weight and viability assessments obtained up to Day 4 postpartum.

The study results included mortalities in a control male (found dead), low, and high dose (both euthanized) female on GD24, and GD23, respectively. Although these deaths were consistent with physiologic stress from failure to litter, test item-related microscopic changes in the kidney and GI tract noted in the high and low dose animal, respectively, may have also contributed to their poor clinical conditions. Reproductive indices affected by the test item were noted at 1000 mg/kg bw/day only and included:

1) reduced gestation and higher mortality indices with 4-fold higher mean post-implantation loss and prolonged gestation length; and

2) increased number of dead pups, markedly lower number of pups at birth and still alive on PPD 4 in comparison to the control group.

There were no other test item-related adverse effects on maternal reproductive indices including total corpora lutea, implantation sites, sex ratio, live birth and malformed pups and gross macroscopic changes. There were no adverse clinical observations for pups from birth to Days 0 to 4 post-partum, no statistically-significant difference for mean pup body weights on Day 0 or 4 post-partum, and no pups found dead (male or female), that were related to the administration of test item. Non-reproductive performance endpoints affected by the test item were noted at the high dose (1000 mg/kg bw/day) only, and included:

1) clinical signs of salivation in both sexes and reduced body weight gain in males;

2) slight reductions in mean platelet (both sexes) and eosinophil (males) counts, increased mean serum alkaline phosphatase and total bile acids with individual increased triglycerides (males), and slightly increased mean prothrombin time (males);

3) increased liver (females) and reduced thymus (males) organ weights (absolute and relative to brain and body weight) compared to controls;

4) minimal microscopic changes in the liver, thymus, and testes in males including: retained spermatids in the testes, hepatic mononuclear infiltrate with or without concurrent hepatocellular single cell necrosis, and lymphoid hypocellularity of the thymus, correlating with lower group mean thymus weights; and,

5) microscopic changes in a single high dose female in the brain, kidney, cecum, and thymus including: minimal to mild poliomalacia, neuronal degeneration, and gliosis involving the hippocampus in the brain, mild renal tubular hyaline droplets and minimal cecum mucosa crypt epithelium hyperplasia/hypertrophy.

After 14 days of recovery, all clinical pathology changes were no longer evident, liver weights in females were still slightly elevated (and males slightly reduced), microscopic changes in all tissues except testes were completely reversed, with incomplete reversal of retained spermatids in testes of recovery males. There were no other test item-related findings, including any effects on FOB or ophthalmoscopic evaluations.

In summary, the daily administration of the test item by oral gavage to male and female Sprague-Dawley rats at dose levels of 100, 300 or 1000 mg/kg bw/day from 2 weeks prior to mating and during the mating period for up to 14 days through to post-partum day 4 in pregnant females, resulted in toxicological findings at the high dose only and included minor reductions in platelets and eosinophils, increases in serum alkaline phosphatase activity and total bile acids, and slightly increased PT. Increased liver weights and reduced thymus weights with microscopic changes in the liver, thymus, and testes of males, and brain, kidney, cecum, and thymus of a single female were noted at the high dose. Microscopic changes reversed completely with the exception of incomplete reversal of retained spermatids in testes. There were adverse effects on male and female fertility, gestation length and post implantation loss and number of live fetuses and pup body weights from birth to PPD 4 at 1000 mg/kg bw/day.

Based on the results of this study, the toxicologic No Observable Adverse Effect Level (NOAEL) for daily oral gavage of the test item in male and female Sprague-Dawley rats was 300 mg/kg bw/day, while the NOAEL for reproductive performance and development of the F1 offspring was also 300 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and guideline study.

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

Combined repeated dose toxicity study with reproduction/developmental Toxicity in rats via oral route

Test item was administerd daily by oral gavage to male and female Sprague-Dawley rats at 3 dose levels for:

1) a 14-day period prior to mating,
2) up to 14-days during mating followed by a 2-week recovery period and
3) during gestation in pregnant females from Gestation Day (GD) 0 (representing successful mating) through to Day 4 post-partum.

Possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Post-Partum Day (PPD) 4 were investigated.

The test item and reference item/vehicle were administered daily, during the dosing period, as shown below:

 

Table 1 Study design

Treatment group

Dose Level (mg/kg bw/d)

Dose Conc. (mg/mL)

Dose Volume (mL/kg)

Number of animals

Main

Recovery**

M

F

M

F

1. Control*

0

0

4

12

12

5

5

2. Test item Low

100

25

12

12

-

-

3. Test item Mid

300

75

12

12

-

-

4. Test item High

1000

250

12

12

5

5

  * Group 1 received the vehicle, corn oil, alone.

** Recovery phase animals not placed for mating.

M = male; F = female

The test item was formulated in corn oil. Males and female Sprague-Dawley rats were 10 weeks old and body weights ranged from 411 to 506 g for males and from 256 to 322 g for females at the start of dosing. Males were dosed for a total of 28 days (inclusive of pre-mating and mating periods), females were dosed during the 14-day premating period, during the mating period for up to 14-days and from GD 0 through to Day 4 postpartum. Main study males were euthanized on Day 29. Main study females were euthanized on PPD 5 and Recovery male and female rats were euthanized on Day 56.

Cage side clinical observations were performed twice daily and a detailed clinical observation was performed on days of body weight recordings. Individual body weight was measured on the day of randomization and weekly through the treatment and recovery period for males. Body weights were performed at randomization, once weekly during the pretreatment and treatment period prior to confirmed mating on GD 0, 7, 10, 14, 17 and 20 and post-partum day (PPD 0 and 4) for females. A Functional Observation Battery (FOB), was performed on 5 animals/sex/group prior to their scheduled necropsy during the last week of dosing (during lactation for females), and during the 14-day recovery period. Clinical pathology (hematology, clinical chemistry, coagulation, and urinalysis) was performed on 5 animals/sex/group prior to their scheduled necropsy. Ophthalmoscopy funduscopic and biomicroscopic (slit lamp) examinations were performed on 5 animals/sex from Groups 1 and 4 towards the end of the dosing period. Estrous cycles were determined prior to and during mating. Dams were observed for parturition at least 3 times a day starting from GD 20, litter size and external sex were determined on PPD 0 and 4 and their offspring (F1) had clinical observations, mortality, body weight and viability assessments obtained up to Day 4 postpartum.

The study results included mortalities in a control male (found dead), low, and high dose (both euthanized) female on GD24, and GD23, respectively. Although these deaths were consistent with physiologic stress from failure to litter, test item-related microscopic changes in the kidney and GI tract noted in the high and low dose animal, respectively, may have also contributed to their poor clinical conditions. Reproductive indices affected by the test item were noted at 1000 mg/kg bw/day only and included:

1) reduced gestation and higher mortality indices with 4-fold higher mean post-implantation loss and prolonged gestation length; and

2) increased number of dead pups, markedly lower number of pups at birth and still alive on PPD 4 in comparison to the control group.

There were no other test item-related adverse effects on maternal reproductive indices including total corpora lutea, implantation sites, sex ratio, live birth and malformed pups and gross macroscopic changes. There were no adverse clinical observations for pups from birth to Days 0 to 4 post-partum, no statistically-significant difference for mean pup body weights on Day 0 or 4 post-partum, and no pups found dead (male or female), that were related to the administration of test item. Non-reproductive performance endpoints affected by the test item were noted at the high dose (1000 mg/kg bw/day) only, and included:

1) clinical signs of salivation in both sexes and reduced body weight gain in males;

2) slight reductions in mean platelet (both sexes) and eosinophil (males) counts, increased mean serum alkaline phosphatase and total bile acids with individual increased triglycerides (males), and slightly increased mean prothrombin time (males);

3) increased liver (females) and reduced thymus (males) organ weights (absolute and relative to brain and body weight) compared to controls;

4) minimal microscopic changes in the liver, thymus, and testes in males including: retained spermatids in the testes, hepatic mononuclear infiltrate with or without concurrent hepatocellular single cell necrosis, and lymphoid hypocellularity of the thymus, correlating with lower group mean thymus weights; and,

5) microscopic changes in a single high dose female in the brain, kidney, cecum, and thymus including: minimal to mild poliomalacia, neuronal degeneration, and gliosis involving the hippocampus in the brain, mild renal tubular hyaline droplets and minimal cecum mucosa crypt epithelium hyperplasia/hypertrophy.

After 14 days of recovery, all clinical pathology changes were no longer evident, liver weights in females were still slightly elevated (and males slightly reduced), microscopic changes in all tissues except testes were completely reversed, with incomplete reversal of retained spermatids in testes of recovery males. There were no other test item-related findings, including any effects on FOB or ophthalmoscopic evaluations.

In summary, the daily administration of the test item by oral gavage to male and female Sprague-Dawley rats at dose levels of 100, 300 or 1000 mg/kg bw/day from 2 weeks prior to mating and during the mating period for up to 14 days through to post-partum day 4 in pregnant females, resulted in toxicological findings at the high dose only and included minor reductions in platelets and eosinophils, increases in serum alkaline phosphatase activity and total bile acids, and slightly increased PT. Increased liver weights and reduced thymus weights with microscopic changes in the liver, thymus, and testes of males, and brain, kidney, cecum, and thymus of a single female were noted at the high dose. Microscopic changes reversed completely with the exception of incomplete reversal of retained spermatids in testes. There were adverse effects on male and female fertility, gestation length and post implantation loss and number of live fetuses and pup body weights from birth to PPD 4 at 1000 mg/kg bw/day. Because these effects were only observed in the High dose, at which also signs of general toxicity were observed, they are considered to be secundary effects due to systemic toxicity apparent at this dose level.

Based on the results of this study, the toxicologic No Observable Adverse Effect Level (NOAEL) for daily oral gavage of the test item in male and female Sprague-Dawley rats was 300 mg/kg bw/day, while the NOAEL for reproductive performance and development of the F1 offspring was also 300 mg/kg bw/day.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on repeated dose toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.