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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-04-11 to 2018-03-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: P 10 wks for males (supplementary males in groups 1 and 3: 12 weeks) 12 wks for females (supplementary females in groups 1 and 3: 14 weeks)
- Weight at study initiation: not reported
- Housing: Macrolon plastic cages
- Diet: pelleted rodent diet provided ad libitum
- Water: tap-water provided ad libitum
- Acclimation period: at least 5 days prior to start of pretest (females) or treatment (males)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 22
- Humidity (%): 43 - 69
- Air changes (per hr): at least 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was dosed undiluted (i.e. without the use of a vehicle). Correction was made for specific gravity (factor: 0.9). No correction was made for purity/composition. On each morning of dosing, the required amount of test item was transferred into a container, flushed with nitrogen and stored at room temperature in the tightly closed container until use. This was done for Groups 2, 3 and 4 separately to minimize the time that the test item was exposed to oxygen in the air during dosing.
For dosing of control Group 1, water (Elix, Millipore S.A.S., Molsheim, France) was used.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No chemical analyses were conducted since the test substance was dosed undiluted (i.e. without the use of a vehicle).
Duration of treatment / exposure:
For Groups 0, 50, 100 mg/kg bw/day, males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females that delivered were treated for 50-55 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at 13-15 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for 41 or 54 days.
For the highest dose group (200 mg/kg bw/day), treatment at the high dose was terminated after 10 days of dosing due to unacceptable high toxicity.
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.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Group 1 (control group): 10 males and 10 females
Group 2 (50 mg/kg bw/day): 10 males and 10 females
Group 3 (100 mg/kg bw/day): 10 males and 12 females
Group 4 (200 mg/kg bw/day): 19 males and 19 females
Control animals:
yes
Details on study design:
- Dose selection rationale: Based on the results of a 14-day dose range finder in which dose limiting effects were noted from 300 mg/kg bw/day onward, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 50, 100 and 200 mg/kg bw/day.

- Rationale for animal assignment: Before initiation of pretest (females) or treatment (males), by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least twice daily from start of treatment onwards up to the day prior to necro psy, detailed clinical observations were made for all animals, immediately after treatment and 1 hour
thereafter (i.e. on the peak period of anticipated effects after treatment).

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to first dosing) 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 AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as gfood/kg body weight/day: Yes; 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 post-coitum and during lactation on PND 1, 4, 7 and 13.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day of scheduled necropsy between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes; isoflurane
- Animals fasted: Yes
- How many animals: 5 animals/sex from Groups 1, 2 and 3 (note: Group 4 was terminated preterm)
- Parameters checked in tables 1 and 2 were examined.

CLINICAL CHEMISTRY: Yes
- Day of scheduled necropsy between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes; isoflurane
- Animals fasted: Yes
- How many animals: 5 animals/sex from Groups 1, 2 and 3 (note: Group 4 was terminated preterm)
- Parameters checked in tables 3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: collected overnight (approximately 15-20 hrs during the night prior to scheduled necropsy)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes; animals were deprived of food but water was available
- Parameters checked in table 4 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed in the morning prior to dosing.
- Dose groups that were examined: 5 animals/sex from Groups 1, 2 and 3 (note: Group 4 was euthanized preterm)
- Battery of functions tested: hearing ability, pupillary reflex, and static righting reflex; fore- and hind-limb grip strength, recorded as the mean of three measurements per animal; and locomotor activity as measured by total movements and ambulations. 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 finer movements like grooming, weaving or movements of the head.

IMMUNOLOGY: No
Sacrifice and pathology:
SACRIFICE
- Male animals: Following completion of the mating period (a minimum of 28 days of dose administration).
- Maternal animals: PND 14-16

GROSS NECROPSY
- Gross necropsy consisted of a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The number of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites. From all animals (including animals that died spontaneously or were sacrificed in extremis), samples of the following tissues and organs shown in Table 5 were collected and fixed in 10% buffered formalin.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 6 were prepared for microscopic examination and weighed, respectively.
Statistics:
The following statistical methods were used to analyse the data:

- If the variables could be assumed to follow a normal distribution, the Dunnett-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 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) in the summary tables. 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

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Rales were noted at 200 mg/kg (in most males and several females, decedents included, on several days) and, less frequently, at 100 mg/kg (in two males and two females, decedents included, on one or two days) and 50 mg/kg (in one female on one day). Additionally, a few of the 200 mg/kg animals that were terminated after 10 treatment days showed gasping, laboured respiration, hunched posture and/or piloerection, mostly at the end of their treatment period and/or on the few treatment-free days thereafter. The latter breathing abnormalities and general clinical signs of toxicity were also noted in most decedents. Piloerection was also noted in one surviving male of the 100 mg/kg group. Salivation seen after dosing among animals treated with the test item, in a dose-related manner, was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. shortly after dosing).
No additional clinical signs of toxicity were noted during the weekly arena observations.
Any other clinical signs noted incidentally 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 and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
Severe toxicity was observed at the high dose, leading to the termination of this dose group. At this dose level, there were eight animals euthanized in extremis (male nos. 38, 42, 43 and 51, female nos. 111, 113, 119 and 123, sacrificed between Days 3-13 of treatment). At 100 mg/kg bw/day, one male (no. 30) was euthanized in extremis on Day 14 of treatment and one female (no. 94) was found dead on Day 11 of treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males and females treated at 200 mg/kg bw/day showed reduced body weight gain (statistically significant in males) during their two-week study period. In males this resulted in statistically significantly reduced body weights at study Days 8 and 15. No treatment-related changes in body weights or body weight gain were noted in rats treated up to 100 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males and females treated at 200 mg/kg bw/day consumed less food than controls throughout their two-week study period. Their food consumption after allowance for body weight showed a similar trend. No treatment-related changes in food consumption before or after allowance for body weight were noted in rats treated up to 100 mg/kg bw/day.
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):
Only data from animals in Groups 1, 2 and 3 (0, 50 and 100 mg/kg bw/day, respectively) were available as the high dose animals (200 mg/kg bw/day; Group 4) had to be euthanized preterm.

Haematology parameters were considered not to be affected by treatment up to 100 mg/kg bw/day. The mean number of total white blood cells was lower at 50 and 100 mg/kg bw/day in both sexes. This was statistically significant for males at 50 mg/kg bw/day and females at 100 mg/kg bw/day. However, as all values remained in the historical control range and in the absence of a dose-response relationship, it was not considered treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Only data from animals in Groups 1, 2 and 3 (0, 50 and 100 mg/kg bw/day, respectively) were available as the high dose animals (200 mg/kg bw/day; Group 4) had to be euthanized preterm.

The mean concentration of urea was statistically significantly lower in males at 100 mg/kg compared to controls (4.2 mmol/L versus 5.2 mmol/L; relative difference: 19%). Group mean value was below the available historical control range. The statistically significant change noted for calcium level in males at 50 mg/kg bw/day was considered unrelated to treatment due to the absence of a dose-related trend.

Urinalysis findings:
no effects observed
Description (incidence and severity):
Only data from animals in Groups 1, 2 and 3 (0, 50 and 100 mg/kg bw/day, respectively) were available as the high dose animals (200 mg/kg bw/day; Group 4) had to be euthanized preterm. Urinalysis parameters were considered not to be affected by treatment up to 100 mg/kg bw/day.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Only data from animals of Groups 1, 2 and 3 (0, 50 and 100 mg/kg, respectively) were available as the high dose animals (200 mg/kg; Group 4) had to be euthanized preterm.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
It was noted that the mean values for total movements and ambulations at 50 mg/kg in males were about 1.5-fold higher compared to the control values. In the absence of a dose-related response, these differences were considered to be unrelated to treatment with the test item.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Only data from animals in Groups 1, 2 and 3 (0, 50 and 100 mg/kg bw/day, respectively) were available as the high dose animals (200 mg/kg bw/day; Group 4) had to be euthanized preterm. There were no test item-related alterations in organ weights up to 100 mg/kg bw/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations for any animal at 50 or 100 mg/kg bw/day. Principal macroscopic findings at 200 mg/kg bw/day related to moribundity consisted of emaciation (one female), distension with gas of (parts of) the gastro-intestinal tract (four males and two females), enlarged or thickened lung (one male and one female) and pale discoloration of the lung (two males and one female). In addition, one female at 200 mg/kg bw/day (no. 113) was noted with discoloration and dark red gelatinous contents of the small intestine. This necropsy observation together with the microscopic findings of esophagus, trachea and/or glandular stomach erosions were indicative for an irritating property of the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Only data from animals in Groups 1, 2 and 3 (0, 50 and 100 mg/kg bw/day, respectively) were available as the high dose animals (200 mg/kg bw/day; Group 4) had to be euthanized preterm. At microscopic examination of the scheduled sacrifices, test item-related findings were noted in the thymus of males of Group 3 (100 mg/kg bw/day), consisting of minimal degrees of lymphoid atrophy (2/5 examined tissues) or increased lymphocytolysis (1/5 examined tissues). The remainder of the recorded microscopic findings of the scheduled sacrifices 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:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone analyses:
Serum levels of T4 in F0 males were considered not to be affected by treatment up to 100 mg/kg bw/day
Details on results:
Mortality:
At 100 mg/kg, one male (no. 30) was euthanized in extremis on Day 14 of treatment and one female (no. 94) was found dead on Day 11 of treatment.
At 200 mg/kg, there were eight animals euthanized in extremis (male nos. 38, 42, 43 and 51, female nos. 111, 113, 119 and 123, sacrificed between Days 3-13 of treatment). Due to this toxicity, the remaining rats of the 200 mg/kg group were no longer dosed from Study Day 11 and were sacrificed a few days later (Study Days 13-15).
Respiratory difficulties (rales, laboured respiration and/or gasping) were noted in all decedents. This was accompanied by hunched posture and/or piloerection in most of them, while a flat posture or chromodacryorrhoea (snout) were noted in one or two decedents.
At macroscopic examination of the 100 mg/kg decedents, no abnormalities were seen in the male while the female found dead showed advanced autolysis. Principal macroscopic findings at 200 mg/kg related to moribundity consisted of emaciation (one female), distension with gas of (parts of) the gastro-intestinal tract (four males and two females), enlarged or thickened lung (one male and one female) and pale discoloration of the lung (two males and one female). In addition, one female at 200 mg/kg (no. 113) was noted with discoloration and dark red gelatinous contents of the small intestine. This necropsy observation together with the microscopic findings of esophagus, trachea and/or glandular stomach erosions were indicative for an irritating property of the test item.
At 100 mg/kg, principal microscopic findings for the two unscheduled deaths were seen in the larynx and consisted of:
- Erosion/necrosis of the epithelium with extension to the underlying tissues, in the male (massive) and the female (moderate).
- Inflammatory cell infiltrate, mainly lymphogranulocytic, in the female (slight).
Principal microscopic findings at 200 mg/kg for the eight unscheduled deaths were seen in the larynx and nasal tissues (in general most obvious in the most caudal levels of the nose):
Larynx:
- Erosion/necrosis of the epithelium with extension to the underlying tissues, in one male (minimal) and three females (marked).
- Inflammatory cell infiltrate, mainly lymphogranulocytic, in three males and four females (up to moderate).
- Luminal exudate, in three females (up to moderate).
- Epithelial hyperplasia, in four males and one female (up to slight).
Nasal tissues:
- Erosions/necrosis of the olfactory epithelium, in four males and one female (up to marked).
- Erosions/necrosis of the respiratory epithelium in four males and one female (up to massive).
- Inflammation, in four males and one female (up to moderate).
- Luminal exudate, granulocytic and/or necrotic and fibrin-like in four males and one female (up to moderate).
- Epithelial metaplasia, in two males and one female (up to moderate).
One female of the control group (no. 67) died at blood sampling prior to scheduled necropsy. As this was a control animal it was not due to treatment with the test item.

Effect levels

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
A combined repeated 28-Day repeated dose toxicity study with the reproduction/developmental toxicity screening according to OECD 422 and in compliance with GLP is available for the test substance. Rats were exposed to 200, 100 and 50 mg/kg bw/day by gavage. Due to high toxicity at 200 mg/kg bw/day the test was terminated for the high-dose group within 10 days. As no relevant effects were observed at 100 mg/kg bw/day a systemic NOAEL of 100 mg/kg bw/day was determined.