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Toxicological information

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:
2015-03-13 to 2016-01-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Jeffcat ZR 50
- Substance type: clear colourless liquid
- Physical state: liquid
- Analytical purity: 97.9%
- Impurities (identity and concentrations): 0.04% water
- Purity test date: no data
- Lot/batch No.: 1203-2014
- Expiration date of the lot/batch: 2016-12-01
- Stability under test conditions: stable for at least twenty-one days
- Storage condition of test material: at room temperature, in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: sixty male and sixty female Wistar Han™:RccHan™:WIST strain rats, obtained from Harlan Laboratories Limited, Oxon, UK
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation: males: 184 to 226 grams, females: 159 to 202 grams
- Fasting period before study: no data
- Housing: in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). Certificates of analysis of the batches of diet were provided.
- Diet (e.g. ad libitum): ad libitum, pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo Research Limited, Oxon, UK) was used.
- Water (e.g. ad libitum): ad libitum, mains drinking water supplied from polycarbonare bottles attached to the cage.
- Acclimation period: nine days, during which time their health status was assessed. The animals were examined for sings of ill-health or injury at receipt.
- The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12, low intensity fluorescent lighting, controlled

IN-LIFE DATES: From: 2015-07-03 to: 2015-10-30

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test item was prepared at the appropriate concentrations as a solution in distilled water.
- The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results showed the formulations to be stable for at least twenty-one days.
- Formulations were prepared weekly and stored at approximately 4 ºC in the dark.
- The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

VEHICLE
- Concentration in vehicle: 0, 1, 2.5 and 7.5 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of test item formulation were taken and analyzed on five occasions for concentration of Jeffcat ZR 50 at Envigo Research Limited, Shardlow, UK, Analytical Services. The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique.
Duration of treatment / exposure:
90 consecutive days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
actual ingested
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
actual ingested
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
actual ingested
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were chosen based on the results of previous toxicity work (provided by the Sponsor)
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: 28 days following termination of treatment
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to thirty minutes post dosing and one hour after dosing. During the treatment-free period, animals were observed daily.
- All animals were examined for overt signs of toxicity, ill-health or behavioral change.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the start of treatment and at weekly intervals thereafter for all non-recovery animals.
- The following parameters were observed: gait, tremors, twitches, convulsions, hyper/hypothermia, skin color, respiration, palpebral closure, bizarre/abnormal/stereotypic behavior, salivation, pilo-erection, exophtalmia, lachrymation, urination, defecation, transfer arousal, tail elevation.

BODY WEIGHT: Yes
- Time schedule for examinations: day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION: yes
- Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and before termination of treatment (during week 12)
- Dose groups that were examined: all non-recovery animals
- Examinations included observation of the anterior structures of the eye following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using a ophthalmoscope was performed.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study (day 90) for all non-recovery animals, at the end of the treatment-free period (day 118) for all recovery group animals.
- Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on days 91 and 119.
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: No
- How many animals: all animals
- Parameters examined: hemoglobin; erythrocyte count; hematocrit; erythrocyte indices:mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration; total leukocyte count; differential leukocyte count: neutrophils, lymphocytes, monocytes, eosinophils, basophils; platelet count; reticulocyte count (slides were prepared but not assessed), prothrombin time was assessed by ‘Innovin’ and activated partial thromboplastin time was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study (day 90) for all non-recovery animals, at the end of the treatment-free period (day 118) for all recovery group animals.
- Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on days 91 and 119.
- Animals fasted: No
- How many animals: all animals
- The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: urea, glucose, total protein, albumin, albumin/globulin ratio (by calculation), sodium, potassium, chloride, calcium, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, total bilirubin, bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the start of the treatment and at weekly intervals thereafter for all non-recovery animals, during week 12 on all non-recovery animals
- Dose groups that were examined: all non-recovery animals
- Functional performances, assessment of sensory ractivity to different stimuli during week 12
- Motor activity: twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Non-recovery animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).
- Forelimb/Hindlimb grip strength: an automated grip strength meter was used. Each nonrecovery animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).
- Sensor reactivity: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests. The following parameters were observed: grasp response, vacolization, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, blink reflex, startle reflex

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- On completion of the dosing period or in the case of recovery group animals, at the end of the treatment-free period all animals were killed by intravenous overdose of a suitable barbiturate followed by exsanguination.
- All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS:
- The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen , testes , thymus, uterus

HISTOPATHOLOGY: Yes
- Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated: adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint) (retained only and not processed), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, epididymides (preserved in modified Davidson's fluid), esophagus, eyes (fixed in Davidson's fluid), gross lesions, heart, ilieum (including Peyer's patches), jejunum, kidneys, liver, lungs (with bronchi) (inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative), lymph nodes (mandibular and mesenteric), muscle (skeletal), ovaries, pancreas, pituitary, prostate, rectum , salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin, spinal cord (cervical, mid thoracic and lumbar), spleen, stomach, testes (preserved in modified Davidson's fluid), thymus, thyroid/parathyroid, tongue (retained only and not processed), trachea , urinary bladder, mammary gland, uterus (with cervix), vagina.
- All tissues from non-recovery control and 75 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed. In
addition, sections of testes from all non-recovery control and non-recovery 75 mg/kg bw/day males were stained with Periodic Acid-Schiff (PAS) stain and examined.
- Since there were indications of treatment-related changes in the liver and spleen, examination was subsequently extended to include similarly prepared sections of the liver and spleen (both sexes) from animals in the low, intermediate and recovery dose groups. In addition, the liver was sectioned at 10μm for one control male, one control female, two non-recovery high dose males and females and two recovery high dose males and females and was stained with Oil Red O (ORO).
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: grip strength, motor activity, body weight change, hematology, blood chemistry, absolute organ weights, body weight-relative organ weights.
Data were analyzed using the decision tree from the Provantis TM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs detected that were considered to be related to test item toxicity. There were isolated incidences of three control males showing fur loss. As the control animals did not recieve any test item, the observation of fur loss was of no toxicological importance. One male treated with 75 mg/kg bw/day showed signs of chromodacryorrhea which later resulted in staining around the eyes. This observation was considered to be an isolated incident and to be of no toxicological importance.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths on study
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- There was an overall reduction in body weight gain in animals of either sex treated with 75 mg/kg bw/day and during weeks 7, 8 and 11, males from this treatment group showed a statistically significant reduction in group mean body weight gains. From week 7, weight gains in these males were generally lower than controls for the remainder of the study. A statistically significant reduction in weight gain during weeks 4 and 8 were evident in females from this treatment group and during week 11, these females showed actual body weight losses.
- During the recovery phase, body weight gains for either sex were comparable to recovery controls.
- No toxicologically significant effects were detected in animals of either sex treated with 10 or 25 mg/kg bw/day.
- Males treated with 25 mg/kg bw/day showed a statistically significant reduction in body weight gain during week 11 whilst females from this treatment group showed a statistically significant reduction in body weight gain during week 8. Body weight gains for the remainder of the study for these animals were comparable to controls therefore in isolation the intergroup differences were considered not to be of toxicological significance.

Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
food conversion efficiency was lower for animals of either sex treated with 75 mg/kg bw/day during Weeks 8 and 11 for males, and Weeks 4, 8 and 11 for females; these reductions correlated with the reduced body weight gains.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following intergroup differences were considered not to be of toxicological significance. Non recovery males from all treatment groups showed a statistically significant reduction in neutrophils. Non recovery females from all treatment groups showed a statistically significant increase in erythrocyte count. A true dose related response was not evident in either sex and the majority of individual values were within background control ranges for both parameters.
There was a statistically significant reduction in mean corpuscular hemoglobin evident in females treated with 75 and 25 mg/kg bw/day. Females treated with 75 mg/kg bw/day also showed a statistically significant reduction in mean corpuscular hemoglobin concentration. The majority of individual values were within background control ranges for both parameters.
Recovery females treated with 75 mg/kg bw/day showed statistically significant increases in erythrocyte count and eosinophils. These females also showed statistically significant
reductions in mean corpuscular hemoglobin and mean corpuscular volume. The majority of individual values were within the background control ranges.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Both non recovery males and females treated with 75 mg/kg bw/day showed a statistically significant increase in alanine aminotransferase and aspartate aminotransferase when compared with controls. In recovery animals at this dose level, a similar effect was evident in these parameters. The majority of individual values were above the background control ranges and correlated with the histopathological findings detected in the liver.
All treated non recovery males showed a statistically significant reduction in total protein. Although the majority of individual values were within background control ranges this reduction can be correlated with the histopathological findings detected in the liver. No such effects were detected in females treated with 25 or 10 mg/kg bw/day.
Males treated with 10 mg/kg bw/day showed a statistically significant increase in glucose levels. The majority of individual values were above the background control ranges. A true dose relationship was not evident and there was no effect on the other treatment groups. Therefore this was considered to be of no toxicological importance. Recovery males treated with 75 mg/kg bw/day showed a statistically significant reduction in phosphorus. In the absence of a similar effect detected in non-recovery males at the end of the treatment period, the intergroup difference was considered of no toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no toxicological significant effects detected in the organ weights measured.
- Non-recovery males from all treatment groups showed a statistically significant increase in group mean thymus weights both absolute and relative to terminal body weight. There was no dose relationship present and the majority of individual values were within the background control ranges. There were no histopathological findings detected in the thymus therefore this was considered to be of no toxicological importance.
- At 75 and 25 mg/kg bw/day, non-recovery males showed a statistically significant increase in kidney weight both absolute and relative to terminal body weight. The majority of individual values were within the background control ranges and a true dose related response was not evident. There were no histopathological findings detected in the kidney therefore this was considered to be of no toxicological importance.
- There were statistically significant increases in both testes and epididymides weights from the non-recovery males treated with 75 mg/kg bw/day. The majority of individual values for each tissue were within the background control ranges and there were no histopathological findings detected. Therefore the intergroup differences were considered to be of no toxicological importance.
- Females treated with 10 mg/kg bw/day showed a statistically significant reduction in liver weight both absolute and relative to terminal body weight. In the absence of a similar effect in males or in females at 25 or 75 mg/kg bw/day, the intergroup difference was considered not to be of toxicological significance.
- Recovery females treated with 75 mg/kg bw/day showed a statistically significant increase in absolute and relative adrenal weight. In the absence of a similar effect detected in nonrecovery females at the end of the treatment period or any associated histopathological correlates, the intergroup difference was considered not to be of toxicological significance.
- Recovery females treated with 75 mg/kg bw/day also showed a statistically significant increase in liver weight both absolute and relative to terminal body weight. Although histopathological changes were evident in these females, the majority of the individual organ weights were within historical control ranges. In the absence of a similar effect in non-recovery females at the end of the treatment period, the intergroup difference was considered to be of limited toxicological significance.
Gross pathological findings:
no effects observed
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All treated male groups showed a statistically significant increase in grip strength values during the final forelimb Test. There was no dose relationship or any associated clinical signs to suggest neurotoxicity. This only occurred in one out of the three tests therefore the intergroup differences were considered to be of no toxicological importance.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Two males and four females treated with 75 mg/kg bw/day, two females treated with 10 mg/kg bw/day and four control females had reddened lungs at necropsy. There were no histopathological findings detected in this tissue and in view of the fact that the finding was also presented in controls, the macroscopic abnormalities were considered to be of no toxicological importance.

LIVER: There was centrilobular hydropic degeneration (presenting as enlarged vacuolated cytoplasm with centrally located nuclei; Oil-Red-O negative for fat) present from minimal to moderate in all non-recovery animals treated with 75 mg/kg bw/day. A mixed inflammatory infiltrate was present in the centrilobular area, minimal or mild in 8/10 males and all females treated with 75 mg/kg bw/day and at a minimal level in 2/10 males treated with 25 mg/kg bw/day. Single cell necrosis (minimal, centrilobular) was present in 7/10 males and 5/10 females treated with 75 mg/kg bw/day and in 2/10 males treated with 25 mg/kg bw/day. After the recovery period centrilobular hydropic degeneration was still apparent in 4/10 males and 2/10 females. Mixed inflammatory infiltrate in the centrilobular area was present in 5/10 males and females. Centrilobular single-cell necrosis at a minimal level was still present in 4/10 males and 5/10 females.

SPLEEN: Vacuolated macrophages were noted at a minimal or mild level in the spleen of 7/10 males and in all females treated with 75 mg/kg bw/day and at a minimal level in one male treated with 25 mg/kg bw/day. After the recovery period, vacuolated macrophages persisted in the spleen of one male and 5/10 females.
Histopathological findings: neoplastic:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No effects were detected in females treated with 25 mg/kg bw/day.
Key result
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effects were detected in males treated with 10 mg/kg bw/day.

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Analytical verification of test item formulation:

The formulations investigated during the study were found to comprise test item in the range of 91 to 102% and, thus, the required content limit of +/- 10% with reference to the nominal content was met.

In addition, the test item was found to be stable in the formulations when kept for 12 and 21 days in the refrigerator (4°C) due to results which met the variation limit of 10% from the time-zero mean.

In conclusion, the results indicate that the accurate use of the test item and distilled water as vehicle during this study. The formulations were found to be homogeneously prepared (visual inspection) and sufficient formulation stability under storage conditions was proven.

Applicant's summary and conclusion

Conclusions:
The oral administration of the test substance to rats by gavage, at dose levels of 10, 25 and 75 mg/kg bw/day resulted in reduced body weight gain at 75 mg/kg bw/day and treatment-related microscopic changes in the liver and spleen in either sex treated with 75 mg/kg bw/day and in males treated with 25 mg/kg bw/day. No such effects were detected in females treated with 25 mg/kg bw/day or in animals of either sex treated with 10 mg/kg bw/day, therefore the ‘No Observed Effect Level’ (NOEL) for females was considered to be 25 mg/kg bw/day and 10 mg/kg bw/day for males. Based on the proposed mechanism of action of osmotic effects and the observation of transient vacuolization in other aliphatic amines, this observation of vacuolization is considered also to be transient and non-adverse and as such the effects are of minimal toxicological importance. Regarding necrosis observed in the liver, the observation was described as “Centrilobular single-cell necrosis at a minimal level”. This description does not meet the criteria of “multi-focal or diffuse” as stated in the ECHA Guidance on the Application of the CLP Criteria for consideration of STOT RE classification. Therefore, these effects do not, by themselves or together, indicate “significant” toxicity in relation to Specific Target Organ Toxicity Repeated Exposure (STOT RE) classification in GHS. Since these observations in the OECD 408 study are considered not to support STOT RE classification, there is no need for this classification.