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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
August 29th to November 19th, 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Reliability of original study is 1
Justification for type of information:
Justification for Read Across is given in Section 13 of IUCLID.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 1996
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
October 2008
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test.
Version / remarks:
July 1995
Qualifier:
equivalent or similar to guideline
Guideline:
other: OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Qualifier:
equivalent or similar to guideline
Guideline:
other: OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents
Version / remarks:
July 2000
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl 3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoate
EC Number:
700-636-5
Cas Number:
5413-49-0
Molecular formula:
C10H1804
IUPAC Name:
Ethyl 3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoate

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. The testing laboratory has general and reproduction/ developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, German.
- Females nulliparous and non-pregnant: yes.
- Age at study initiation: approximately 11 weeks.
- Weight at study initiation: 307-310 g (males), 199-203 (females).
- Housing: during the pre-mating animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm). During the mating period females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage; females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the lactation pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: free access to tap-water.
- Acclimation period: at least 5 days prior to start of treatment.

DETAILS OF FOOD AND WATER QUALITY: diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 18-24 °C.
- Humidity: 40-70 %.
- Air changes: 15 room air changes/hour.
- Photoperiod: 12-hour light/12-hour dark cycle.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test substance and the vehicle. No correction was made for the purity/composition of the test substance.

DOSE VOLUME: 5 ml/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted after completion of the in-life phase (a validated method was not available during the in-life phase), according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110 % of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10 %. Formulations were considered stable if the relative difference before and after storage was maximally 10 %.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females were exposed for 44-50 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 43 (Group 1), 67 and 70 (Group 3) and 79 (Group 4) were not dosed during littering.
Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
group 4
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a 10-day dose range finding study, the dose levels for this combined 28-day oral gavage study with reproduction/ developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg.
- Rationale for animal assignment: prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20 % of the sex mean.

Examinations

Observations and examinations performed and frequency:
The following observations and examinations were performed on the parental animals. 5 animals/sex/group were selected for functional observations, locomotor activity, clinical pathology, macroscopic examination, organ weights and histopathology.

MORTALITY/VIABILITY: Yes.
- Time schedule: at least twice daily.
- Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstance of any death was recorded in detail.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals, at least immediately (0-15 minutes) after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.
- The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

BODY WEIGHT: Yes
- Time schedule for examinations: males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
- Time schdedule: weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Yes.
- Time schedule for examinations: subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: blood samples were collected immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Collection of blood samples: blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with EDTA for haematological parameters (0.5 ml), with citrate for clotting tests (0.45 ml) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 ml). An additional blood sample (0.25 ml) was collected into untreated tubes for determination of bile acids.
- Anaesthetic used for blood collection: Yes, by using isoflurane.
- Animals fasted: Yes. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was provided.
- How many animals: collected from the selected 5 animals/sex/group.
- Parameters checked: white blood cells, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: blood samples were collected immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Collection of blood samples: blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with EDTA for haematological parameters (0.5 ml), with citrate for clotting tests (0.45 ml) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 ml). An additional blood sample (0.25 ml) was collected into untreated tubes for determination of bile acids.
- Anaesthetic used for blood collection: Yes, by using isoflurane.
- Animals fasted: Yes. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was provided.
- How many animals: collected from the selected 5 animals/sex/group
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate, bile acids.

FUNCTIONAL OBSERVATIONS
The following tests were performed on the selected 5 animals/sex/group: hearing ability, pupillary reflex, static righting reflex, grip strength, score 0 = normal/present, score 1 = abnormal/ absent, locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head. During motor activity monitoring, all animals were caged individually (pups were housed in their home cages and kept warm using bottles filled with warm water).
The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling). These tests were conducted after observation for clinical signs (incl. arena observation, if applicable) at no specific time point, but within a similar time period after dosing for the respective animals.
During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
Sacrifice and pathology:
NECROPSY: all males and the selected 5 females/group were deprived of food overnight (with a maximum of approximately 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetized using isoflurane and subsequently exsanguinated. Necropsy was conducted on the following days:
Females which delivered- Lactation Days 5-7.
Females which failed to deliver1 (no. 41) - 22 days after the last day of the mating period (females without evidence of mating).
Males - Following completion of the mating period (a minimum of 28 days of dose administration).
Euthanized in extremis (no. 50) - When pain, distress or discomfort was considered not transient in nature or was likely to become more severe.

GROSS PATHOLOGY: Yes. All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

COLLECTION OF ORGANS AND TISSUES: Yes. Samples of the following tissues and organs were collected and fixed in 10 % buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands): adrenal glands, aorta, brain - cerebellum, mid-brain, cortex, caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, epididymides, eyes (with optic nerve (if detectable) and harderian gland), female mammary gland area, femur including joint, heart, ileum, jejunum, kidneys, lacrimal gland, exorbital, larynx, liver, lung, infused with formalin, lymph nodes - mandibular, mesenteric, esophagus, nasopharynx, ovaries, pancreas, peyer's patches [jejunum, ileum] if detectable, pituitary gland, preputial gland, prostate gland, rectum, salivary glands - mandibular, sublingual, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord -cervical, midthoracic, lumbar, spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid if detectable, tongue, trachea, urinary bladder, uterus, vagina, all gross lesions. Nasopharynx, esophagus, aorta, pancreas, salivary glands-mandibular, sublingual, skin and tongue were not examined by the pathologist since no signs of toxicity were noted at macroscopic examination.
For all the remaining animals, female which failed to deliver: cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, vagina, all gross lesions. Nasopharynx, esophagus, aorta, pancreas, salivary glands-mandibular, sublingual, skin and tongue were not examined by the pathologist since no signs of toxicity were noted at macroscopic examination.
For all the remaining animals, female which failed to deliver: cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, vagina, all gross lesions.

HISTOPATHOLOGY
A peer review on the histopathology data was performed by a second pathologist. The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 to examine staging of spermatogenesis.
- The preserved organs and tissues of the animals of all dose groups were killed in extremis.
- All gross lesions of all animals (all dose groups).
- Thymus and spleen of all selected 5 females and stomach, testes and epididymides of all selected 5 males of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
- The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina) of all males that failed to sire and all females that failed to deliver healthy pups (female no. 41 (not mated), and female no. 50 (assessment of the mammary gland area, since most pups of the litter had no milk visible in the stomach during first and last litter check.) with male no. 10 (female sacrificed due to difficult parturition).
- Histotechnology: all the above organ and tissue samples, were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin. From the selected 5 males of the control and high dose group, and all males suspected to be infertile or which died before mating, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin.

ORGAN WEIGHTS: terminal body weight was recorded from all males and the selected 5 females/sex/group. The following organ weights were recorded from the following animals on the scheduled day of necropsy: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles including coagulating glands, spleen, testes, thymus, thyroid including parathyroid, uterus (including cervix) (for the selected 5 animals/sex/group); epididymides, testes (for all the remaining animals)
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores). Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.
Salivation seen after dosing among the 100, 300 and 1000 mg/kg dose groups during treatment was considered to be a physiological response rather than a signof systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to taste of the test substance.
Incidental findings that were noted included scabs, alopecia and rales. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed,these were considered signs of no toxicological relevance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test substance.
One control female (no. 50) was sacrificed in extremis due to a difficult parturition.
Description (incidence and severity):
No toxicologically relevant changes in body weights and body weight gain were noted. Body weight and body weight gain of males at 1000 mg/kg was lower than control throughout the treatment period, achieving a level of statistical significance on most occasions. Given the slight nature of this change, this was considered to be of no toxicological relevance.
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were noted.
The higher food intake (before or after correction for body weight) for females at 1000 mg/kg between Days 0-4 of the post-coitum phase was of a temporary and slight nature, and therefore considered to be of no toxicological relevance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Description (incidence and severity):
No toxicologically relevant changes occurred in haematological parameters of treated rats.
The degree of delay in prothrombin time (PT) of males at 1000 mg/kg was slight in nature and occurred in the absence of supportive changes in the liver, and was therefore considered to be of no toxicological relevance.
Description (incidence and severity):
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
Any (statistically significant) changes in clinical biochemistry parameters were considered to be of no toxicological significance as they occurred in the absence of a dose-related trend and supportive morphological changes, and/or remained within the range considered normal for rats of this age and strain. These changes consisted of higher alanine aminotransferase activity (ALAT) in males at 1000 mg/kg (only slightly exceeding the normal range), higher aspartate aminotransferase activity (ASAT) in males at 300 and 1000 mg/kg, higher alkaline phosphatase activity (ALP) in males at 100, 300 and 1000 mg/kg (not statistically significant, but means at 1000 mg/kg slightly outside normal range), lower total protein level in males and females at 300 and 1000 mg/kg, lower albumin level in males at 1000 mg/kg and females at 300 and 1000 mg/kg, higher total bilirubin levels in females at 100, 300 and 1000 mg/kg, lower glucose level in males at 100 mg/kg, lower sodium level in females at 1000 mg/kg, higher potassium level in females at 1000 mg/kg (not statistically significant), and lower calcium level in females at 100, 300 and 1000 mg/kg.
Urinalysis findings:
not examined
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals. No toxicologically significant changes in motor activity were noted. Mean counts for total movements and ambulations during the first interval of 5 minutes appeared higher for males at 300 and 1000 mg/kg. Since these means appeared similar to control levels during the second interval, and group mean of all intervals combined was similar across the groups, no toxicological relevance was ascribed to this change. All remaining male and female groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period.
Immunological findings:
not examined
Description (incidence and severity):
No toxicologically relevant changes were noted in organ weights and organ to body weight ratios.
Any statistically significant changes in organ weights were considered not to be a sign of toxicity since means remained within the range considered normal, a clear dose-related trend was absent, and no morphological correlates were found. These changes consisted of a lower heart weight in males at 100, 300 and 1000 mg/kg, lower adrenal weight in males at 1000 mg/kg and lower spleen weight in males at 100 and 1000 mg/kg. The higher kidney to body weight ratio of males at 1000 mg/kg was ascribed to the lower terminal body weight, since absolute kidney weights were normal.
Description (incidence and severity):
Necropsy did not reveal any toxicologically relevant alterations. The incidence of necropsy findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance.
Neuropathological findings:
not examined
Description (incidence and severity):
There were no treatment-related microscopic findings.
All microscopic findings recorded were within the normal range of background pathology encountered in Wistar (Han) rats of this age. No abnormalities were seen in the reproductive organs of the rats which could account for their infertility or parturition problems. The spermatogenic staging profiles were normal for all examined males treated of the control and 1000 mg/kg group.
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Analysis of dose preparations

- Accuracy of preparation: the concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90 % and 110 %). No test substance was detected in the Group 1 formulation.

- Homogeneity: the formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10 %).

- Stability: formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours.

Applicant's summary and conclusion

Conclusions:
NOAEL (male/female) ≥ 1000 mg/kg/ day (actual dose received)
Executive summary:

The substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 44-50 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 5 hours at room temperature.

Generally, sporadic, statistically significant changes were noted in some parameters in the study. None of these were considered as toxicologically significant. No parental toxicity was observed up to the highest dose level tested (1000 mg/kg). No toxicologically significant changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

In conclusion, treatment with the test substance by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed no parental toxicity or reproduction and developmental toxicity up to 1000 mg/kg.

Based on these results, a parental NOAEL of at least 1000 mg/kg was derived.