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Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 April 2015 - 20 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
performed under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
(Sept 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
(May 2008)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
(August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2012 by MHLW (0331 No.7), METI (No. 5) and MOE (No. 110331009)
Deviations:
no
Principles of method if other than guideline:
The exposure control analysis was based on the following guidelines:- Guideline on Bioanalytical Method Validation, European Medicines Agency (EMA), EMEA/CHMP / EWP/192217/2009, 21 July 2011;- Guidance for industry: Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER) and Center for Veterinary Medicine (CVM), May 2001.
GLP compliance:
yes (incl. QA statement)
Remarks:
dd. 03 November 2015
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reaction Mass of CXN1-55
IUPAC Name:
Reaction Mass of CXN1-55
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): CXN 1-55- Substance type: Organic- Physical state: Off-white to brownish solid- Storage condition of test material: In refrigerator (2-8°C) desiccated

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
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 6 weeks- Weight at study initiation: 140-166g (males), 118-141g (females) (main study), 154-155g (males), 132-133g (females) (dose range finding study)- Fasting period before study: Animals were only deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm), except during locomotor activity monitoring, when animals were housed individually in a Hi-temp polycarbonate cage- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum (except during locomotor activity monitoring, when no food was available and overnight before sacrifice (maximum 24 hours))- Water: tap water, ad libitum (except during locomotor activity monitoring, when no water was available)- Acclimation period: At least 5 daysENVIRONMENTAL CONDITIONS (set conditions)- Temperature (°C): 18-24- Humidity (%): 40-70- Air changes (per hr): at least 10- Photoperiod (hrs dark / hrs light): 12/12IN-LIFE DATES: From: 07 April 2015 To: 20 October 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No correction was made for the purity/composition of the test substance.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Accuracy, homogeneity and stability were determined for formulations prepared for in week 1, week 6 and week 13 in duplicate. For determination of accuracy, samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations. For determination of stability, additional samples were taken at 50% height and stored at room temperature under normal laboratory light conditions for 6 hours. The volumetric flasks were filled up to the mark and extracted with methanol. The shaking time was 15 seconds. The extracts were further diluted to obtain an end solution of methanol and concentrations within the calibration range. Analyses were conducted according to a validated method for sodium bromide and triethanolamine.The accuracy of preparation was considered acceptable if the mean measured concentrations are 90-110% of the target concentration for solutions, or 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation is ≤ 10%. Formulations were considered stable if the relative difference before and after storage is maximally 10%.
Duration of treatment / exposure:
At least 90 days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
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:
3 females (dose range finding study);10 (main study)5 (recovery groups, control and high dose groups only )
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on a 14-day range finding study (for details see below)- Recovery groups were included for control and high dose groups. The animals in these groups were left untreated for 8 weeks before sacrifice.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes- Time schedule: At least twice dailyDETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: At least once daily from start of treatment onwards. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena.FUNCTIONAL OBSERVATIONS:During week 12 of treatment, the following tests were performed on the first 5 animals/sex/group after dosing at no specific time point, but within a similar time period after dosing for the respective animals:- hearing ability, pupillary reflex (L/R), static righting reflex;- fore- and hind-limb grip strength (recorded as the mean of three measurements);- motor activity test (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system)- total movements and ambulations BODY WEIGHT: Yes - WeeklyFOOD CONSUMPTION: Yes- WeeklyFOOD EFFICIENCY:- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes WATER CONSUMPTION- Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.OPHTHALMOSCOPIC EXAMINATION: Yes- Time schedule for examinations: at pretest and week 13- Dose groups that were examined: all (including recovery groups)HAEMATOLOGY: Yes- Time schedule for collection of blood: At the end of the treatment (all animals, including recovery groups) and at the end of recovery period (recovery groups only)- Anaesthetic used for blood collection: Yes (isoflurane)- Animals fasted: Yes, animals were deprived of food overnight (for a maximum of 24 hours), but water was available.- How many animals: all animals- Parameters: according to guidelinesCLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: At the end of the treatment (all animals, including recovery groups) and at the end of recovery period (recovery groups only)- Anaesthetic used for blood collection: Yes (isoflurane)- Animals fasted: Yes, animals were deprived of food overnight (for a maximum of 24 hours), but water was available.- How many animals: all animals- Parameters: according to guidelinesURINALYSIS: No OTHER:- Recovery females for control and high dose groups, and the first 5 females for low and mid dose groups had a daily lavage from day 70 up to and including day 90 to determine the stage of estrous;- Blood levels of bromide and triethanolamine were analysed in all animals at the end of the treatment (including rats in recovery group) and at the end of the recovery period (recovery animals only)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, according to guidelines (including organ weight determination)HISTOPATHOLOGY: Yes, according to guidelines
Other examinations:
For all males, the following assessments were performed:- Sperm samples were taken from the proximal part of the vas deferens (right):1. Sperm motility and progressive motility were assessed from all main and recovery samples;2. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm froma differential count of at least 200 spermatozoa (if possible) per animal was recorded.Evaluation was performed for all samples of the main control and main high dose group. Evaluation of samples of the low and mid dose and recovery groups was not performed, as no treatment-related effect was seen at the end of treatment.One testis and one epididymis (left) were removed, and kept in the freezer. After thawing the left testis and epididymis were weighed, homogenized and evaluated for sperm numbers.
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.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. 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, non-treatment-related
Description (incidence and severity):
No clinical signs of toxicity were noted at up to and including 1000 mg/kg bw/day. Incidental findings (including alopecia, a wound in the neck, scabs in the neck or on one or both cheeks and a broken tail) occurred within the range of background findings and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment. The weekly arena observations did not reveal any clinical signs in addition to those noted at the daily observations.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls throughout the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control animals.
Food efficiency:
no effects observed
Description (incidence and severity):
No effects were observed on food efficiency.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No effects were observed on water consumption.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental findings in week 13 included focal corneal oedema or opacity, pinpoint corneal opacities and haemorrhage in the retina. The nature and incidence of these findings were within the range considered normal for rats of this age and strain.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in haematology parameters of treated rats. Statistically significant differences noted between control and treated animals (reduction of relative lymphocyte count in males at 100 mg/kg bw/day (-2.4% compared to controls) and increased mean corpuscular volume (MCV) in males at highest dose (+2.9% compared to controls)). Related to the minor level of these effects and since these observations did not show a dose-related trend they were considered incidental findings and not to represent changes of biological relevance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. Statistically significant differences noted between control and treated males consisted of decreased alanine transferase (ALAT) levels at high dose in males (-22.7% compared to controls), decreased total bilirubin in males (-12.5%, -8.3% and -16.6% compared to controls for rats exposed to respectively 100, 300 and 1000 mg/kg bw/day, statistically significant only at low and high dose). Furthermore, glucose levels were decreased for exposed males (-13.0%, -6.4% and -22.8% compared to controls for rats exposed to 100, 300 and 1000 mg/kg bw/day respectively, statistically significant only at low and high dose). In high dose males, cholesterol levels and bile acid levels were statistically significantly decreased (with -17.4% and -38% respectively). In high dose females, statistically significant alterations were noted for protein level (+7.4%), albumin concentration (+8.4%) and creatinine concentration (- 4%). These effects were considered not to be toxicologically relevant as they remained within the normal range for rats of this age and strain and/or occurred in the absence of a treatment-related distribution.Plasma levels of chloride were dose-dependently increased in all treated rats (with 2.9%, 13% and 26.0% for males and 1.9%, 6.7% and 21.0% for females treated at 100, 300 and 1000 mg/kg bw/day respectively. Chloride was measured using an Ion Selective Electrode (ISE). It is known that bromide, a constituent of the test substance, can interfere with this measurement, resulting in falsely increased chloride results (Dimeski et al. Clinica Chimica Acta, 411 (2010)). Exposure control analysis showed that treated rats had elevated plasma levels of bromide. Therefore, the apparent increase in chloride was considered to be an artifact resulting from interference by test substance-derived bromide with the ISE and not considered an adverse effect. This rationale was further substantiated by the fact that in the recovery groups, chloride (and bromide) levels of animals exposed to 1000 mg/kg bw/day were identical to those of control rats.For a number of clinical biochemistry parameters (mainly aspartate aminotransferase, alanine aminotransferase and bile acids) increased values were noted in individual females treated at 300 or 1000 mg/kg bw/day. These findings were considered not to be of toxicological relevance because they occurred in only a few animals and in the absence of corroborative histopathological changes.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were not adversely affected by treatment. A statistically significantly lower forelimb grip strength noted in females treated at 1000 mg/kg bw/day remained well within the normal range for rats of this age and strain and was considered not to represent a change of biological relevance. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period. In the absence of a treatment-related distribution, the statistically significantly lower number of total movements noted in females at 1000 mg/kg bw/day was considered not to be related to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In females, absolute and relative liver weights were increased in high dose females (16.7% and 11.6%, statistically significant). Furthermore, statistically significant increase was observed in females for absolute and relative kidney weights (absolute: 25.9% at 1000 mg/kg bw/day, relative: 9.2% and 20% at 300 and 1000 mg/kg bw/day respectively). Since the statistically significantly higher kidney weight in females at 300 mg/kg bw/day was only present in the relative to body weight ratio and within historical background values and therefore considered not to be treatment-related. After an 8-week treatment-free period, the liver and kidney weights of females of the 1000 mg/kg bw/day group were within normal range. Other findings in females included decrease of absolute and relative thymus weight (absolute: respectively -12.0%, -16.8% and -13.0% for 100, 300 and 1000 mg/kg bw/day, only statistically significant at mid dose level; relative: respectively -11.2%, -14.0% and -16.8% for 100, 300 and 1000 mg/kg bw/day, only statistically significant at high dose level)). It should be noted that for both males and females the effect on thymus ameliorated after eight weeks recovery period (difference to control in males respectively -5.9% and -8.3% for absolute and relative weight, and in females respectively -6.7% for both absolute and relative weight). No adverse effects were seen at organ weights (absolute and relative) for treated males. The heart weight of males treated at 1000 mg/kg bw/day was statistically significantly decreased, but since this was only seen for absolute weight and the effect was relatively small (-10.8%), this was not found to be adverse.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related gross observations. Incidental findings among control and treated animals were 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 unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related adverse microscopic findings were present in reproductive organs of females starting at 300 mg/kg bw/day. A decrease/absence of corpora lutea in the ovaries in two females at 300 mg/kg bw/day. In the uterus of the female with absent corpora lutea at 300 mg/kg bw/day, slight inflammation of the mucosa was seen, furthermore light luminal debris, moderate squamous metaplasia were seen. This female was found to have luminal dilation and endometrial glands were found to be missing. One additional female at 300 mg/kg bw/day was found to have squamous metaplasia (minimal degree). Cycle stage was found to be abnormal in respectively 5/10 and 7/10 females at 300 and 1000 mg/kg bw/day (no match between ovary, uterus and vagina histology). Vaginal inflammatory cell infiltrate was seen in 1 female at 300 mg/kg bw/day (minimal) and in 7 females at 1000 mg/kg bw/day (5 minimal, 2 slight). Furthermore, luminal debris consisting of desquamated epithelium, granulocytes and/or bacteria in the vagina was seen in 4 females at 300 mg/kg bw/day (minimal) and in 7 females at 1000 mg/kg bw/day (2 minimal, 3 slight and 2 moderate).Additional test item-related findings were found at 1000 mg/kg/day. These consisted of hypertrophy/hyperplasia of the follicular cells of the thyroid glands, observed at increased incidence (10/10) and severity (mean severity/organ of 1.6) in males treated at 1000 mg/kg/day compared to incidences of 6/10, 8/10 and 5/10 and mean severities of 1.3, 1.1 and 1.0 in groups treated at respectively 0, 100 and 300 mg/kg bw/day. Inflammatory cell infiltrate was observed at increased incidence (6/10) in males treated at 1000 mg/kg bw/day, compared to incidences of 1/10 in the other groups of males. Degeneration/regeneration of Harderian glands was observed at increased incidence (6/10) and/or severity (up to slight) in males treated at 1000 mg/kg bw/day, compared to incidences of 2/10 or 3/10 and a severity of mostly minimal in the 100 and 300 mg/kg/day treated males of the main study. Inflammatory cell infiltrate in the adrenal glands was observed at increased incidence in 5/10 females treated at 1000 mg/kg bw/day (minimal degree). There were no test item-related findings noted after a 8-week treatment-free period.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
ESTROUS CYCLE EXAMINATIONEstrous cycle as determined by vaginal lavage was regular in all females examined, except for two females treated at 300 mg/kg bw/day which had irregular cycles. A somewhat higher frequency of metestrus phase was noted in two females treated at 1000 mg/kg bw/day. The irregularities of the estrous cycle in these females were corroborated by histopathological evidence of disturbances in the normal reproductive cycle and were thus considered to be related to treatment.SPERM PARAMETERSSperm motility, morphology and count were not affected by treatment. At end of treatment, a low number of sperm cells was noted for one high dose male. As the sperm count of the testes was normal, this was considered a chance finding and not related to treatment. In addition, a low percentage of sperm motility and progressive sperm motility was noted for two other high dose males. As this was noted in 2/10 males and in absence of corroborative findings, this was considered not toxicologically relevant. Moreover, at end of recovery, the percentage of progressive sperm motility in all high dose males was lower than at end of treatment, and this was also noted for the concurrent control males. A statistically significantly higher epididymal sperm count at 1000 mg/kg bw/day at the end of the treatment period was considered not to be related to treatment due to the direction of the change and the absence of corroborative morphological alterations in the epididymides.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: At 300 mg/kg bw/day and above: Abnormalities in estrous cycle (coinciding with microscopic alterations in ovaries and/or uterus and/or vagina).
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects were noted up to and including the highest tested dose (1000 mg/kg bw/d) in males.

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
female reproductive system
Organ:
ovary
uterus
vagina
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Exposure control:

Bromide and triethanolamine levels were found to be below detection limit in the blood of control animals. Bromide levels for exposed males (females) were found to vary between 174-218 μg/mL (189-203 μg/mL), 569-636 μg/mL (424-531 μg/mL) and 1970-1970 μg/mL (1940-1730 μg/mL) for rats exposed to 100, 300 and 1000 mg/kg bw/day respectively. At the end of the recovery period, bromide levels were below detection.

Analysis of formulations (main study):

A small response at the retention time of the test substance was observed in the control formulations. It was not considered to derive from the formulation since a similar response was obtained in the analytical blanks. The concentrations analysed in the formulations of low, mid and high dose groups prepared for use in week 1 and week 6 and in the formulation of the low dose group prepared for use in week 13 were in agreement with target concentrations (i.e. mean accuracies between 100% and 107%). For the formulation of mid and high dose groups prepared for use in week 13, the mean accuracy was slightly above the target concentration (i.e. 113% and 112% of target). Since the accuracies are only slightly above the criterion, it is considered to have an negligible impact on the study outcome. The formulations of low and high dose groups were homogeneous (i.e. coefficient of variation ≤ 3.3%).

Applicant's summary and conclusion

Conclusions:
In a 90-day oral repeated dose toxicity study with rats, conducted according to OECD/EC guidelines and GLP principles, abnormalities in estrous cycle (coinciding with microscopic alterations in ovaries and/or uterus and/or vagina) were observed in females dosed at 300 mg/kg bw/day and above. In males, no adverse effects were observed up to and including the highest tested concentration of 1000 mg/kg bw/d. Based on these data, the overall NOAEL of CXN1-55 was found to be 100 mg/kg bw/day for females and 1000 mg/kg bw/d for males.
Executive summary:

A 90-day oral repeated dose toxicity study was conducted with CXN1-55 according to OECD/EC guidelines and GLP principles. Male and female rats were exposed to 0, 100, 300 or 1000 mg/kg bw/day via oral gavage. The study included a control and a high dose recovery groups, for which the treatment phase was followed by an eight weeks recovery period before sacrifice. Chemical analysis of the formulations confirmed accuracy of dosing. No mortality occurred during the study. No clinical signs of toxicity were noted at up to and including 1000 mg/kg bw/day. Hearing ability, pupillary reflex, static righting reflex and grip strength were not adversely affected by treatment. Body weights and body weight gain of treated animals remained in the same range as controls throughout the study. Absolute and relative food consumption were not affected by test substance exposure. No treatment-related ocular changes were observed. No toxicologically relevant changes occurred in haematology and clinical biochemistry parameters of treated rats.

In females, absolute and relative liver and kidney weights were increased at 1000 mg/kg bw/day, but these returned to normal range after the recovery period. Furthermore in females decrease of absolute and relative thymus weight at 300 and above was seen. This effect was ameliorated after eight weeks recovery period. No adverse effects were seen at organ weights (absolute and relative) for treated males. Sperm motility, morphology and count were not affected by treatment. Estrous cycle was found to be irregular for two females treated at 300 mg/kg bw/day. Histopathology revealed test item-related adverse microscopic findings in reproductive organs of females starting at 300 mg/kg bw/day. In males, increased incidence and severity of hypertrophy/hyperplasia of the follicular cells of the thyroid glands at 1000 mg/kg bw/day was observed but was considered not to be adverse.

Taken all data together, the overall NOAEL of CXN1-55 was found to be 100 mg/kg bw/day for females and 1000 mg/kg bw/d for males.