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Administrative data

Description of key information

In a repeated dose oral toxicity study combined with a reproduction/developmental toxicity screening study with rats (OECD 422), adverse histopatological effects were observed in the thyroid gland of females at 300 and 1000 mg/kg bw/day. Therefore parental NOAEL was established to be at least 100 mg/kg bw/day based on the effects in the thyroid gland.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

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:
4 Jul 2018 - 06 Nov 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000)
Version / remarks:
July 2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: other guidance as listed under Principles of method if other than guideline
Principles of method if other than guideline:
In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 2016);
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch 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, Germany
- Age at study initiation: Approximately 10-11 weeks (males) and 13-14 weeks (females)
- Weight at study initiation: 275 - 314 g (males) or 192 - 223 g (females)
- Fasting period before study: no
- Housing: Pretest (females only) and pre-mating period: group housed (up to 5 animals of the same sex and same dosing group together in polycarbonate cages Macrolon, MIV type, height 18 cm). Mating phase: males and females were cohabitated on a one-to-one basis in Macrolon plastic cages (MIII type, height 18 cm). Post-mating phase: males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed. Lactation phase: pups were housed with the dam (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). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage enrichment, bedding material, food and water.
- Animal enrichment: For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- 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: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
The actual daily mean temperature during the study period was 20 to 22 °C with an actual daily mean relative humidity of 51 to 78 %.

IN-LIFE DATES: From: 04 Jul 2018 to 07 Sept 2018
Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
corn oil
Details on oral exposure:
Method of formulation: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Initially and based on the stability data, the dosing formulations were prepared at room temperature and protected from light in daily portions.

Storage conditions of formulations: If not used on the day of preparation, the formulations were stored in the refrigerator up to a maximum of eight days during the first two weeks of dosing and up to a maximum of 4 days during the remaining dosing period. On the day of use, the dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. All formulations were used for dosing within 6 hours after preparation, if used on the day of preparation, or within 6 hours after taken out of the refrigerator. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

Justification for use and choice of vehicle (if other than water): Vehicle was selected based on trial experiments performed at the testing facility and based on information provided by the sponsor.

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 performed using a validated analytical procedure. Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15 % for suspensions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10 %. Stability analyses performed previously in conjunction with the method development and validation study. The results demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination.
Females were exposed for 50-65 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 41-51 days. Females were not dosed if they were littering at the moment of dosing.
Frequency of treatment:
Once daily, 7 d/w
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High dose
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: The dose levels were selected based on the results of a 10-day dose range finder with oral administration of the test item in rats, and to determine the peak effect of occurrence of clinical signs after dosing. No guidelines were applicable as this study was intended for dose level selection purposes only. Test system, procedures and techniques were identical to those used during the main study. Three females were dosed with 500 and 1000 mg/kg bw/day, by once daily oral gavage for 10 consecutive days. The dose levels were selected based on information provided by the sponsor (Acute oral toxicity study in rats: LD50 > 2000 mg/kg bw; no deaths and no signs of toxicity observed up to 2000 mg/kg bw). Mortality was checked twice daily throughout the study. Clinical observations were done at least daily from Days 1-10, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing. Body weights were recorded on day 1 prior to dosing and on days 5 and 10 after dosing. Food consumption was monitored over days 1-5 and 5-10. All animals were subjected to an external, thoracic and abdominal examination on Day 11 (scheduled necropsy). Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy. No organs were fixed and histopathological examination was not performed. No mortality or clinical signs were observed during the dose range finder study. Based on the results of the dose range finder, selected dose levels for the main study were 100, 300 and 1000 mg/kg bw/d. Since no clinical signs were observed in the dose range finder, it was decided to perform the clinical observations and functional observations directly after dosing in the main study.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
Yes
- Twice, daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
- During the dosing period, these observations were at least performed directly after dosing based on the (absence of) clinical signs observed in the dose range finder.
- The time of onset, grade and duration of any observed signs were recorded using grades from 1 to 4, where 1 is slight and 4 is very severe. For certain signs, 2 scores were used: absence (grade 0) or presence (grade 1).

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 PND 1, 4, 7, and 13.

FOOD CONSUMPTION:
Yes. Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:
No.

WATER CONSUMPTION:
Yes, on regular basis throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled necropsia.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted:
Males: Yes, overnight (with a maximum of 24 hours). Water was provided.
Females: no
- Number of animals: 5 animals/sex/group
- Parameters checked: according to test guidelines.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled necropsia.
- Animals fasted:
Males: Yes, overnight (with a maximum of 24 hours). Water was provided.
Females: no
- Number of animals: 5 animals/sex/group
- Parameters checked: according to the guidelines.

THYROID HORMONES
- Measurement of total T4 and TSH was conducted for F0 males and females, and PND 14-16 pups.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Yes
- Time schedule for examinations: During Week 4 of treatment (males) and during the last week of lactation (females, i.e. PND 9-12). After dosing, after completion of clinical observations.
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, and fore- and hind-limb grip strength (recorded as the mean of three measurements per animal) and locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system).
Total movements and ambulations were reported.
Deviations: Yes
- Locomotor activity test was performed twice for females as one female escaped during the first measurement.
Sacrifice and pathology:
SACRIFICE
- Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
- F0 males were fasted overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. F0 females were not fasted.
Scheduled euthanasias as follows:
- Males (which sired and failed to sire): after completion of mating period, minimum 28 days of administration.
- Females which delivered: PND 14-16
- Females which failed to deliver: Post coitum day 24 (with evidence of mating), 24 days after the last day of mating period (without evidence of mating)

GROSS PATHOLOGY: Yes
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
- The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

ORGAN WEIGHTS
- On selected 5 animals/sex/group: according to guidelines.
- All remaining animals: epididymis, prostate, seminal vesicles including coagulation glands, testes, thyroid.

HISTOPATHOLOGY: Yes
On selected 5 animals/sex/group in control and high-dose groups and all animals that were euthanized in extremis: according to guidelines.
- On selected 5 males in all groups and all males that failed to sire: additional testes slides.
- On all animals: all gross lesions.
- All remaining animals, including males that failed to sire and females that failed to deliver pups: Cervix, epididymis, glands (coagulation, mammary, parathyroid, pituitary, prostate, seminal vesicle, and thyroid), gross lesions/masses, ovaries, testes, uterus, vagina.
Other examinations:
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage (performed daily until evidence of copulation was observed or termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.
Statistics:
All statistical tests were conducted at the 5 % significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5 % levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: Low dose group vs. control, Mid dose group vs. control, High dose group vs. control.
Parametric: Datasets with at least 2 groups (the designated control group and 1 other group) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric: Datasets with at least 2 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
Incidence: an overall Fisher’s exact test was used to compare all groups at the 5 % significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Piloerection was observed in a single female at 300 mg/kg bw/day dose group and among a few females at 1000 mg/kg bw/day during late post coitum and/or early lactation phase. Based on the incidence and time of occurrence these clinical signs were considered to be related to their pregnancy and maternal care, rather than being treatment related. The persistence of the piloerection in one female for a prolonged period was likely corresponding to the high mortality in its litter. Other clinical signs observed are considered incidental findings, i.e. scabs (control and 100 mg/kg bw/day), alopecia, hunched posture and red tears (chromodacryorrhoea; 1000 mg/kg bw/day). 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 not considered to be signs of toxicological relevance. Salivation seen after dosing among animals of the 300 and 1000 mg/kg bw/day dose group from week 2-3 of the treatment period was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Mortality:
no mortality observed
Description (incidence):
One female of the 1000 mg/kg bw/day group was euthanized on lactation day 3, as she had a total litter loss.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The statistically significant increases observed for body weight gain in the 300 and 1000 mg/kg bw/day dose group females at lactation day 13, when compared to controls, were considered to be the result of relatively low body weight gain in the control group (and also in the 100 mg/kg bw/day dose group) (Historical control data females period 2015-2018: Body weight gain (%) lactation Day 13: mean: 14, P5-P95: 5.7-21.3, n=789). Since the body weight gain in the 300 and 1000 mg/kg bw/day dose group was within the normal range the statistical significances were a fortuitous finding and not related to treatment. The body weight values of two females (one on lactation day 7 and one on lactation day 13) were considered unrealistic and are probably weighing errors, based on the absence of corroborating clinical signs of ill health that might have explained the sudden body weight loss. Moreover, the terminal body weight for one female was in line with was to be expected based on its body weights during the lactation phase.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, the increase in food consumption during post coitum days 4-11 and the higher food consumption after correction for body weight during post coitum days 4-7 were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment and not of toxicological relevance.
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
Description (incidence and severity):
For males, the following changes in haematology were found: Decreased white blood cell count at 100 and 300 mg/kg bw/day (16 %, 19 %, respectively), decreased number of lymphocytes at 300 mg/kg bw/day (32 %), decreased haematocrit values at 100 mg/kg bw/day (5 %). Relative changes in mean values relative to the control group are indicated between parentheses. In absence of a dose response relationship, the alterations in haematological parameters were considered unrelated to administration of the test item. For females, no changes in haematological parameters were noted.

A decrease in prothrombin time was noted in males of 9 % compared with control at 100 mg/kg bw/day, which was considered unrelated to administration of the test item due to the minimal magnitude of the change and in absence of a dose response. In females, a reduction (not statistically significant) in activated partial thromboplastin time (APTT) appeared of 14 % compared with the control at 1000 mg/kg bw/day, which was considered unrelated to treatment as statistical significance was not reached and values were within normal range (Historical control data females period 2015-2018: Activated partial thromboplastin time: mean: 17.6; P5- P95: 13.1-22.7; n=136).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In absence of a dose response relationship, the statistical significances apparent for calcium levels in males treated at 300 mg/kg bw/day and for alkaline phosphatase and potassium levels in females treated at 100 mg/kg bw/day, in comparison with controls, were considered to have occurred by chance and not related to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals.
Motor activity was similar between treated and control groups. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
The mean motor activity in control-group females was relatively low in comparison with the normal range for females of this age and strain (Total movements: mean: 3372, P5- P95: 1529-5618, n=400. Ambulations: mean: 817, P5-P95: 317-1440, n=400) and a large variation in motor activity was observed between the females in the low dose group (100 mg/kg bw/day). Two low dose females showed relatively high motor activity, for total movements as well as the ambulations, during the 7th to 10th (out of twelve) 5-min interval in comparison with the other three low dose females, resulting in a high standard deviation to the mean total movements and ambulation values for the low dose females. The results of the motor activity in the mid and high dose females, i.e. 300 and 1000 mg/kg bw/day respectively, were in the range as expected from the historical control data. In the absence of a dose response relationship and since all group mean values were within the normal range, the motor activity in females was considered not affected by treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant higher liver weights were noted in the 100, 300 and 1000 mg/kg bw/day group females.
One 100 mg/kg bw/day treated female had an abnormal papillary process of the liver (dark red discoloration and hardened), which was grown together with the stomach. This correlated microscopically with marked necrosis, some hemorrhage and a rim of granulation tissue around this lobe and was considered as an incidental finding. Since this was the only female of this group with an apparent higher individual liver weight, the significant liver weight change of the 100 mg/kg bw/day group was considered to be directly related to this single animal and unrelated to treatment with the test item.
The higher liver weights observed in 300 and 1000 mg/kg bw/day females were regarded as test item-related.
Any other differences were considered not to be test item-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Watery fluid in the uterus, found in two females of dose group 300 mg/kg bw/day is related to a stage in the estrous cycle and is a normal finding.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the liver and thyroid gland of the 300 and/or 1000 mg/kg bw/day group females. In the liver, hepatocellular hypertrophy was present at minimal degree in the 1000 mg/kg bw/day treated females. In the thyroid gland, an increased incidence and/or severity (up to mild) of follicular cell hypertrophy was present in 300 and 1000 mg/kg bw/day treated females. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Thyroid hormone analyses: Mean serum T4 levels of F0 males and F0 females were decreased by treatment at 100, 300 and 1000 mg/kg bw/day to a same degree in each sex when compared with the respective controls. The mean T4 levels were approximately 65 % lower in treated males (decreases of 63 %, 64 % and 6 8%, respectively) and approximately 35 % lower in treated females (decreases of 35 %, 38 % and 36 %, respectively), achieving statistical significance at all dose levels in each sex in comparison with controls. In treated males, the group mean values for T4 were below the historical control range at all dose levels tested (mean: 4.41, P5- P95: 2.76-6.34, n=333). In control females, a relatively high mean value for T4 of 3.55 μg/dL was observed in comparison with the historical mean value of 2.61 μg/dL (mean: 2.61, P5- P95: 1.56-4.040, n=48). However, because of the consistency of the decreases in T4 levels and a similar variation between the individual values in each treated group, the changes in T4 levels were considered to be treatment related, despite the fact that the absolute T4 levels in treated females were within the historical control range. The differences between the T4 levels of control and treated females within the current study were comparable to the difference between the values of the mean and lower limit of the historical control range (i.e. approximately 40 % from the mean). Mean serum TSH levels were considered not to be affected by treatment and no indication of a feedback response to the decreased T4 levels was apparent. Several relatively high TSH values were observed, but were not accompanied by low T4 levels in these individual animals. Furthermore, the distribution of these high TSH values over the dose groups also did not indicate a relation to treatment, but did affect the group mean values and corresponding standard deviation for this parameter.
Details on results:
In this study, a marked reduction of total T4 was observed in parental male and female rats and in PND 14-16 male and female pups at all three dose level of 100, 300 and 1000 mg/kg bw/day. Therefore, a no observed effect level (NOEL) could not be established in this study and was considered to be below the lowest dose level of 100 mg/kg bw/day. Furthermore, the possible impact of the changes in T4 could not be assessed within this type of screening study and was therefore not taken into account when determining the parental and developmental NOAEL.
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Analysis of dose preparations:


In the control group formulation, no test substance was detected. The concentrations analysed in the formulations of groups exposed to 100, 300 and 1000 mg/kg bw/day were in agreement with target concentrations (i.e. mean accuracies between 85 % and 115 %). The formulations of the low and high dose group were homogeneous (i.e. coefficient of variation ≤ 10 %).

Conclusions:
In a repeated dose oral toxicity study combined with a reproduction/developmental toxicity screening study with rats (OECD 422), adverse histopatological effects were observed in the thyroid gland of females at 300 and 1000 mg/kg bw/day. Therefore parental NOAEL was established to be at least 100 mg/kg bw/day based on the effects in the thyroid gland.
Executive summary:

A repeated dose oral toxicity study combined with a reproduction/developmental toxicity screening study with rats was performed according to OECD 422/EC guidelines and GLP principles. The test item was administered by daily oral gavage to male and female rats at dose levels of 100, 300 and 1000 mg/kg bw/day. 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 50-65 days). In parental female rats, dose-related changes were observed in the liver at 1000 mg/kg bw/day, comprising increased hepatic weights and correlating hepatocellular hypertrophy, and in the thyroid gland at 300 and 1000 mg/kg bw/day, i.e. follicular cell hypertrophy. Corresponding changes were not observed up to the highest dose level in male rats. Furthermore, serum total T4 levels were approximately 65 % lower in males and approximately 35 % lower in females at all three dose levels when compared to controls. The findings in the liver were observed in female rats at 1000 mg/kg bw/day and were considered non-adverse. A linear relationship between the effects in the thyroid gland and the decreased T4 levels was not apparent in females. Effects on the thyroid gland in males were absent. In addition, treatment-related changes in TSH level were not observed in both sexes, and thus it is unlikely that the changes in thyroid gland and T4 levels were the result of a normal physiological feedback-regulation in the thyroid hormone homeostasis. In the worst case, the effects on the thyroid should be considered representative of an adverse effect, because they seemed not to be the result of a normal physiological feedback-regulation in the thyroid hormone homeostasis. The impact of the changes in T4 levels could not be evaluated within this type of screening study, because it is unknown if the decrease in total T4 (comprising free and protein-bound T4) had any effect on the metabolic active, free T4 concentration, which is responsible for the feedback-regulated thyroid homeostasis. Based on the histopatological effects observed in the thyroid gland, a parental no observed adverse effect level (NOAEL) for the test item was established to be 100 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
OECD Guideline under GLP conditions
System:
endocrine system
Organ:
liver
thyroid gland

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

A repeated dose oral toxicity study combined with a reproduction/developmental toxicity screening study with rats was performed according to OECD 422/EC guidelines and GLP principles. The test item was administered by daily oral gavage to male and female rats at dose levels of 100, 300 and 1000 mg/kg bw/day. 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 50-65 days). In parental female rats, dose-related changes were observed in the liver at 1000 mg/kg bw/day, comprising increased hepatic weights and correlating hepatocellular hypertrophy, and in the thyroid gland at 300 and 1000 mg/kg bw/day, i.e. follicular cell hypertrophy. Corresponding changes were not observed up to the highest dose level in male rats. Furthermore, serum total T4 levels were approximately 65 % lower in males and approximately 35 % lower in females at all three dose levels when compared to controls. The findings in the liver were observed in female rats at 1000 mg/kg bw/day and were considered non-adverse. A linear relationship between the effects in the thyroid gland and the decreased T4 levels was not apparent in females. Effects on the thyroid gland in males were absent. In addition, treatment-related changes in TSH level were not observed in both sexes, and thus it is unlikely that the changes in thyroid gland and T4 levels were the result of a normal physiological feedback-regulation in the thyroid hormone homeostasis. In the worst case, the effects on the thyroid should be considered representative of an adverse effect, because they seemed not to be the result of a normal physiological feedback-regulation in the thyroid hormone homeostasis. The impact of the changes in T4 levels could not be evaluated within this type of screening study, because it is unknown if the decrease in total T4 (comprising free and protein-bound T4) had any effect on the metabolic active, free T4 concentration, which is responsible for the feedback-regulated thyroid homeostasis. Based on the histopatological effects observed in the thyroid gland, a parental no observed adverse effect level (NOAEL) for the test item was established to be 100 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 does not require classification for STOT RE according to Regulation (EC) No 1272/2008 (CLP).