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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 September 2016 - 02 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

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:
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:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
dd 13 February 2017
Limit test:
no

Test material

Constituent 1
Reference substance name:
adipic acid dihydrazide
IUPAC Name:
adipic acid dihydrazide
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: at room temperature- Stability under test conditions: stable- Solubility and stability of the test substance in the solvent/vehicle: 102 g/L at at 20°C in water. Stability in 1% aqueous carboxymethyl cellulose for at least 5 hours at room temperature and 8 days in the refrigerator and 3 weeks in the freezer (≤ -15°C) was confirmed over the concentration range 1 to 200 mg/mL.OTHER SPECIFICS: No correction was made for purity/composition of the test item.

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS- Source: Charles River Deutschland, Sulzfeld, Germany.- Females (if applicable) nulliparous and non-pregnant: yes- Age at study initiation: ca. 6 weeks- Weight at study initiation: males 143-176 g, females 104-144 g- Fasting period before study: no- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material and paper as cage-enrichment. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum- Water: tap water, ad libitum- Acclimation period: At least 5 days DETAILS OF FOOD AND WATER QUALITY: Diet, water, bedding and cage enrichment 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 (°C): mean daily 21-22- Humidity (%): mean daily 44-66- Air changes (per hr): at least 10- Photoperiod (hrs dark / hrs light): 12/12IN-LIFE DATES: From: 15 September 2016 To: 02 February 2017

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
This study should provide a rational basis for toxicological risk assessment in man. The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1%
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily prior to dosing, and were homogenized to a visually acceptable level. No correction was made for purity/composition of the test item. VEHICLE - Justification for use and choice of vehicle (if other than water): based on trial formulations performed at Charles River Den Bosch and on information from the Sponsor - Amount of vehicle (if gavage): 5 mL/kg bw. Actual dose volumes were calculated weekly according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analyses of formulations were conducted during the study to assess accuracy and homogeneity. Samples of formulations were taken for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations, in Weeks 1, 6 and 13)
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily, 7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
0
Remarks:
Concurrent vehicle controls
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:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were based on results of a 14-day oral range finding study with Adipic acid dihydrazide (ADH) by daily gavage in the rat
Positive control:
Not applicable.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes - Time schedule: at least twice daily for viability and mortalityDETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: at least once daily after dosing. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. BODY WEIGHT: Yes- Time schedule for examinations: weeklyFOOD CONSUMPTION AND COMPOUND INTAKE: yes- Time schedule for examinations: weeklyFOOD EFFICIENCY: noWATER CONSUMPTION AND COMPOUND INTAKE: subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.OPHTHALMOSCOPIC EXAMINATION: Yes - Time schedule for examinations: pretest and week 13- Dose groups that were examined: pretest: all animals, week 13: controls and high-dose groupHAEMATOLOGY: Yes- Time schedule for collection of blood: week 13 prior to sacrifice- Anaesthetic used for blood collection: Yes (isoflurane)- Animals fasted: Yes, overnight for a maximum of 24 hours- How many animals: all- Parameters examined: white blood cells, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, reticulocytes, red blood cell distribution width (RDW), haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentrations, platelets; prothrombin time, activated partial thromboplastin time.CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: week 13- Animals fasted: Yes, overnight for a maximum of 24 hours- How many animals: all- Parameters examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium , potassum, chloride, calcium, inorganic phosphate, bile acidsURINALYSIS: No NEUROBEHAVIOURAL EXAMINATION: Yes - Time schedule for examinations: week 13 - Dose groups that were examined: all, first 5 animals/sex/group- Battery of functions tested: sensory activity (hearing, pupillary reflex, static righting reflex) / grip strength (fore- and hinb-limb) / motor activity IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. The following organ weights were recorded: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus including cervix, prostate, seminal vesicles including coagulating glands, thyroid including parathyroid.HISTOPATHOLOGY: YesThe following slides were examined by a pathologist: all tissues according to the guidelines, collected at the scheduled sacrifice from all controls and high-dose animals, sciatic nerve, thymus, pancreas, liver, spleen, kidney and brain of all males and females of low- and mid-dose animals, based on (possible) treatment-related changes in these organs in high dose group; all gross lesions.
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) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Piloerection was observed in all 1000 mg/kg bw/day males and in one 1000 mg/kg bw/day female, however its occurrence was very brief and transient. Hunched posture and lean appearance were seen in a single 1000 mg/kg male for a short period of time. No findings were noted during the arena observations in this study.Salivation was briefly seen after dosing among a few 1000 mg/kg females. This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity. Any other clinical signs noted during the treatment period 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 did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative body weight were significantly reduced in 1000 mg/kg bw/day males in a statistically significant manner from Week 8 and Week 5, respectively, until the end of treatment. A statistically significant reduction in percentage body weight gain was also seen in 1000 mg/kg bw/day females (throughout the study), however this reduction was not as robust as in male counterparts.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A slight reduction in food consumption (absolute) was observed in 1000 mg/kg bw/day males during the second half of the study, when compared to controls. In females, food consumption remained similar to controls over the study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The nature and incidence of ophthalmology findings noted during pretest and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in haematological parameters were observed:• Haemoglobin and haematocrit levels were reduced in 1000 mg/kg bw/day males and females. • Mean cell volume of red blood cells (MCV), was reduced in 1000 mg/kg bw/day females.• Mean cell haemoglobin concentration (MCHC) was reduced in 1000 mg/kg bw/day males and females at 300 and 1000 mg/kg bw/day.• Red blood cell counts were reduced in 1000 mg/kg bw/day males (not statistically significant in females).• Mean cell haemoglobin mass (MCH) was reduced in 1000 mg/kg bw/day females.• Percentage red blood cell distribution width (% RDW) was increased in both male and female 1000 mg/kg bw/day animals.• Relative monocyte levels were increased in female 1000 mg/kg bw/day animals.•Analysis of clotting parameters revealed increased prothrombin time (PT) in 1000 mg/kg males.Any other statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in clinical biochemistry parameters were observed:• Urea and bile acid levels were increased in 1000 mg/kg bw/day males and females, when compared to controls. The increase in bile acid levels was particularly robust. • Alanine aminotransferase (ALAT) levels were reduced in 300 and 1000 mg/kg bw/day females. • Glucose levels were decreased in male 1000 mg/kg bw/day animals and female 300 and 1000 mg/kg bw/day animals• Mean aspartate transaminase (ASAT) and mean alkaline phosphatase (ALP) levels were not statistically significantly altered, however in two individual animals of 1000 mg/kg bw/day group, ASAT and ALP levels were much higher than controls.• Albumin and total bilirubin levels were also affected, with increases in albumin levels seen in 100 and 1000 mg/kg bw/day males and 300 mg/kg bw/day females, while total bilirubin levels were increased in 1000 mg/kg bw/day males. • Chloride levels were reduced in 1000 mg/kg bw/day males and females.• Potassium levels were increased in 1000 mg/kg bw/day females, calcium levels were increased in 300 and 1000 mg/kg bw/day females and inorganic phosphate levels were increased in 1000 mg/kg bw/day males and females.Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
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. Foregrip strength was similar between control and high dose animals. A statistically significant reduction in hindgrip strength was seen in 300 mg/kg bw/day females only, however this effect occurred in a non-dose related manner, and was therefore not considered as toxicologically relevant.Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
the following test item-related changes were observed:- higher liver weights (absolute and relative to body weights) in females at 1000 mg/kg bw/day (statistically significant, 42% higher than in controls); statistically significantly higher relative liver weights were observed in 300 mg/kg bw/day females (10% above the controls).- statistically higher relative liver weights in 1000 mg/kg bw/day males (29% above controls)- statistically higher relative kidney weights in males and females at 300 and 1000 mg/kg bw/day (resp. 10% and 8% above the controls in m/f at 300 mg/kg bw/day and 42% and 29% above the controls in m/f at 1000 mg/kg bw/day)- lower thymus weight (absolute and relative to body weights) in males and females (not statistically significant in females) at 1000 mg/kg bw/day. Other organ weight differences were statistically significant when compared to the control group but were considered to be the result of a test item-related effect on final body weight or within the normal range of the organ weights in rats of that strain and age.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related macroscopic findings were observed at 1000 mg/kg bw/day and consisted of:• reduced size of the thymus in 5/10 males and 8/10 females. • pale discoloration of the liver in 7/10 females. • reduced size of the thyroid gland in 2/10 females and 1/10 males and pale discoloration in 4/10 females and 1/10 males. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
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 of males starting at 300 and females at 1000 mg/kg bwd/ay, in the kidney of females starting at 300 mg/kg bw/day and males at 1000 mg/kg bwday and in brain, sciatic nerve, pancreas, thymus and spleen in the 1000 mg/kg group bw/day males and females.In the liver, an increased severity of inflammatory cell infiltrates was observed in males (moderate degree) at 1000 mg/kg bw/day; hepatocellular microvesicular vacuolation and multifocal hepatocellular necrosis was present up to slight and/or moderate degree in males and females treated at 1000 mg/kg bw/day; an increased severity of single cell necrosis (minimal single cell necrosis was considered to be the background level) was observed in males treated at 300 mg/kg bw/day.In the brain, vacuolation of neuronal tissue in the cerebellar peduncle area was observed up to moderate degree in few males and up to slight degree in a few females treated at 1000 mg/kg bw/day.In the sciatic nerve, demyelination was observed up to moderate degree in almost all males and up to slight degree in a few females treated at 1000 mg/kg bw/day.In the kidney, tubular hypertrophy at the outer medullary was observed at minimal degree in a single female treated at 300 mg/kg bw/day and up to slight degree in all males and females treated at 1000 mg/kg bw/day.In the pancreas, degranulation of acinar cells was observed up to slight degree in males and females treated at 1000 mg/kg bw/day.In the thymus, lymphoid depletion was observed up to slight degree in few males and a single female treated at 1000 mg/kg bw/day.In the spleen, an increased severity of extramedullary hematopoiesis and pigmentation up to moderate degree was observed in males and females treated at 1000 mg/kg bw/day.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
Details on results:
Other secondary findings which may be reflective of the hepatic alterations included hypoglycemia in 1000 mg/kg bw/day males and females, as well as the increases in albumin levels and PT time. Reduced body weight in 1000 mg/kg bw/day males and females, reduced food intake in 1000 mg/kg bw/day males and clinical signs such as the occasional piloerection, hunched posture and lean appearance were also accompanying the adverse microscopic findings described.Non-adverse tubular hypertrophy was present in a single female at 300 mg/kg bw/day and in males and females at 1000 mg/kg bw/day. Increased kidney weight and changes in clinical biochemical parameters such as urea and ion levels (chloride, calcium, potassium, inorganic phosphates) in 300 and/or 1000 mg/kg bw/day animals may be secondary to this effect.Findings in the pancreas (degranulation acinar cells), thymus (lymphoid depletion, accompanied by reduced thymus weight) and spleen (increased severity of extramedullary hematopoiesis and pigmentation) of 1000 mg/kg bw/day males and females were also considered to be non-adverse. The slight haematological alterations such as reduced haemoglobin and hematocrit levels, mean red cell volume, mean cell haemoglobin counts, mean cell haemoglobin mass, and reduced red blood cell counts in 1000 mg/kg bw/day animals may be accompanying the changes observed in the spleen.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
brain
liver
other: sciatic nerve
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

No test item was detected in the control formulation (Week 1, Week 6 and week 13).

The concentrations analyzed in the formulations of low and mid-dose groups (week 1, 6 and 13) were in agreement with the target concentrations for solutions (i.e. mean accuracies week 1: 104.3% (n = 6) and 102.8% (n = 2), respectively, week 6: 98.7% (n = 6) and 98.3% (n = 2), respectively; week 13: 106.8% (n = 6) and 88.7% (n = 2), respectively), with the exception of the mid-dose group samples prepared on 31 January 2017, which showed a mean accuracy of 88.7%. This deviation was not considered to have affected the integrity of the study outcome, due to its slight nature, its occurrence in mid-dose group (high-dose group formulations were compliant), and the fact that accuracy on other weeks fell within the required concentrations.

The concentrations analyzed in the formulations of high-dose group (Week 1, 6 and 13) were in agreement with the target concentrations for suspensions (i.e. mean accuracies 105.6, 97.1 and 94.2%, respectively, n = 6 in all cases)).
The formulations of low- and high-dose groups (week 1, 6 and 13) prepared were homogeneous (i.e. coefficient of variation week 1: 0.8% and 1.1%, respectively; week 6: 1.2% and 1.3%, respectively; week 13: 1.9% and 9.4%, respectively (n = 6)).

Applicant's summary and conclusion

Conclusions:
In a GLP-compliant OECD guideline 408 study with rats the NOAEL was set at 100 mg/kg bw/day, based on adverse changes in liver at the next dose levels (increased severity in single cell necrosis, changes in clinical liver parameters, weight changes).
Executive summary:

In a GLP-compliant OECD guideline 408 study, groups of 10 male and female rats were administered the test substance in 1% CMC by oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day for 90 days. There were no mortalities. Clinical signs were limited to transient piloerection, in all high dose males and one high-dose females, hunched posture and lean appearance in a single high-dose male for a short period of time and brief salivation after dosing in a few high-dose females. Absolute and relative body weights were significantly reduced in 1000 mg/kg bw/day males from weeks 8 and 5, respectively, towards the end of the treatment; statistically significantly reduced body weight gain was observed in 1000 mg/kg bw/day females. Adverse treatment-related effects were identified in the brain and sciatic nerve of 1000 mg/kg animals, and in the livers of animals treated at 300 and 1000 mg/kg bw/day (dose-related). Examination of the neuronal tissue revealed vacuolation of tissue in the brain at the level of the cerebellar peduncle and the demyelination of the sciatic nerve in few animals of the 1000 mg/kg bw/day treatment group. Analysis of clinical signs, motor activity and functional observations did not reveal any changes that may correlate with the abovementioned neurological alterations. Liver findings in 1000 mg/kg bw/day males and females included the combination of hepatocellular changes (microvesicular vacuolation, increased inflammatory cell infiltrates and the degenerative process of multifocal necrosis) together with elevated clinical liver parameters (alanine aminotransferase, aspartate transaminase and alkaline phosphatase in single males, total bilirubin in males and bile acids in males and females) and higher liver weight, which in combination are considered to be adverse. In males treated at 300 mg/kg bw/day, single cell necrosis in the liver was also considered to be adverse. Here, adversity was based on the increased severity (slight in 300 mg/kg bw/day males, compared to minimal in control males), changes in clinical liver parameters, and the fact that an increase in single cell necrosis probably precedes the presence of multifocal necrosis, which occurred in the 1000 mg/kg bw/day group. Other secondary findings which may be reflective of the hepatic alterations described above include hypoglycemia in 1000 mg/kg bw/day males and females, as well as the increases in albumin levels and PT time. Tubular hypertrophy was present in a single female at 300 mg/kg bw/day and in males and females at 1000 mg/kg bw/day, which was considered to be non-adverse Increased kidney weight and changes in clinical biochemical parameters such as urea and ion levels (chloride, calcium, potassium, inorganic phosphates) in 300 and/or 1000 mg/kg bw/day animals may be secondary to this effect. Findings in the pancreas (degranulation acinar cells), thymus (lymphoid depletion, accompanied by reduced thymus weight) and spleen (increased severity of extramedullary hematopoiesis and pigmentation) of 1000 mg/kg males and females were also considered to be non-adverse. Based on the adverse findings observed in the liver of 300 and 1000 mg/kg bw/day animals and in the brain and sciatic nerve of 1000 mg/kg bw/day animals, a No Observed Adverse Effect Level (NOAEL) of 100 mg/kg bw/day bw/day was established.