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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 March 2014 - 13 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Details on test material:
- Physical state: solid
- Analytical purity: 100%
- Expiration date of the lot/batch: not supplied (retest after 2 years)
- Storage condition of test material: room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: 10-11 weeks old
- Weight at study initiation: males 264 to 321g; females 198 to 237g
- Fasting period before study: no
- Housing: Initially, all non-recovery animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the non-recovery animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. Recovery group animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding.
- Diet: ad libitum. A pelleted diet (Rodent 2018C Teklad
Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used.
- Water: ad libitum. Mains water.
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): >=15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 05 June 2014 (first day of treatment) and 28 July 2014 (final day of necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP. Since the ICP-MS method used for formulation analysis was considered not to indicate stability, test item formulation stability was not determined and therefore, fresh formulations were prepared each day and dosed within three hours of preparation. It is assumed that the formulation was stable for this duration. As stability was not
determined, this is an exception with regards to GLP and has been reflected in the GLP compliance statement. Homogeneity of the test item formulations was determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Arachis oil used as test item is not soluble in water
- Concentration in vehicle: 16.7, 50 or 167 mg/mL
- Amount of vehicle (if gavage): 6 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) using an external standard technique.
Analytical procedure
Preparation of standard solutions
Stock solutions of test item in tetrahydrofuran were prepared for external standard calibration. An aliquot, 100 mg, of test item was accurately weighed into a 100 mL volumetric flask and brought to volume with tetrahydrofuran to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in 2% nitric acid with a concentration of 0.05, 0.1 and 0.15 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.
Analysis of samples
The formulations received were extraced with tetrahydrofuran. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with tetrahydrfuran. This was then ultra-sonicated for 30 minutes and centrifuged at 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with 2% nitric acid to achieve the working concentration.
Preparation of accuracy samples
Samples of arachis oil BP were acurately fortified with known amounts of the test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis as th etest samples described above.
Preparation of linearity standards
A range of standard solutions were prepared in tetrahydrofuran from a stock solution of 1.003 mg/mL by serial dilution covering the concentration range 0 to 0.15350 mg/mL.
Instrumental setup
The standards and samples were analysed by ICP-MS using the following conditions:
ICP-MS: Agilent 7500 cx
Acquisition mode: Spectrum (multi-tune)
Element and Mass: Zn, 66
Peak Pattern: Full quant (3)
Repetitions: 5
Interference Equation: None
Detector Setting: Auto
Integration Time: 0.3 second

Results
Specificity
The control dose samples and an analysed solvent blank showed no significant interfering response at the retention time fo the test item. The standard solutions contained a peak specific for the test item whose area changed accordingly with the known concentration; hence the specificity of the method by retention time was confirmed.
Linearity
The linearity of the analytical system used for sample analyses was found to have a quadratic correlation within the calibration range of 0 to 0.15 mg/mL. The R2 fit of the calibration curve to the data was 0.997 and was considered to be acceptable.
Accuracy
The fortified samples of Arachis oil BP were found to have arecovery value of +/-10% of the fortification.
Test item formulations
The formulations investigated during the study were found to comprise test item in the range of 86% to 143 %.
Duration of treatment / exposure:
Day 1 to day 42
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Non-recovery dose groups: 12 males/12 females per dose
Recovery dose groups: 5 males/5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses selected based on the results of the 14-day range-finding study
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups.
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately after dosing, 30 minutes and one hour after dosing. During the treatment-free period recovery animals were observed daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately after dosing, 30 minutes and one hour after dosing. During the treatment-free period recovery animals were observed daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Recovery animals were weighed on Day 1 (prior to dosing) and then weekly until termination.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD 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: Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: N/a
- Dose groups that were examined: N/a

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 42 for males; Day 4 post partum for females (non-recovery/control groups). Day 56 for recovery group animals (i.e. after 14 day recovery period).
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: No recovery/control groups - 5 males/5 females; Recovery group - all animals
- Parameters included in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 42 for males; Day 4 post partum for females (non-recovery/control groups). Day 56 for recovery group animals (i.e. after 14 day recovery period).
- Animals fasted: No
- How many animals: No recovery/control groups - 5 males/5 females; Recovery group - all animals
- Parameters included in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: final week of treatment/final week of recovery
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters included in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to start of treatment and at weekly intervals thereafter.
- Dose groups that were examined: All non-recovery groups
- Battery of functions tested: sensory activity / grip strength / motor activity:

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (full internal and external examination)
HISTOPATHOLOGY: Yes (see table 4)
Other examinations:
Organ weights
Statistics:
See below

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths on the study. There were no clinical signs considered to be related to the toxicity of the test item. During the treatment period, sporadic instances of increased post-dose salivation were seen in animals of either sex given the test item at a dose level of 1000 mg/kg bw/day from Day 5 of dosing. Increased post-dose salivation was also seen in one male given the test item at 300 mg/kg bw/day on Day 28 and some control animals on Day 29 of dosing. Such observations are common in this type of study and are normally associated with the taste of the formulation. Male 5 from the control group was observed with a scab from Day 27, which may have resulted from infighting and was considered unrelated to dosing.
There were no clinical signs for any of the recovery animals during the treatment-free period.
BODY WEIGHT AND WEIGHT GAIN
There was no effect of treatment at any dose level on male body weight development during the treatment or treatment-free periods.
There was no effect of treatment on body weight development in females during the pre-pairing, gestation or lactation phases of the study. Group mean body weight gains in treated females allocated to the recovery groups were generally similar to the respective controls throughout the study.
FOOD CONSUMPTION AND FOOD EFFICIENCY
Food intake and food efficiency across all groups of test item treated males remained similar to controls throughout the study.
There was no effect of treatment with the test item at any dose level on food consumption or food efficiency in females during the pre-pairing phase of the study. Food intake for treated females at all dose levels during gestation and lactation remained similar to controls. Food intake and food efficiency in treated recovery females were also similar to the respective controls throughout the study.
WATER CONSUMPTION
Visual inspection of water bottles did not indicate any differences in water intake for treated animals of either sex in comparison with controls.
OPHTHALMOSCOPIC EXAMINATION
Not applicable
HAEMATOLOGY
No toxicologically significant effects were detected in animals of either sex at any dose level.
At the end of the treatment period, there were no statistically significant differences for hematology parameters in treated animals of either sex when compared with controls.
At the end of the recovery period, males previously given the test item at 1000 mg/kg bw/day showed marginally but statistically significantly higher group mean values of hemoglobin (p<0.05), red blood cells (p<0.05) and hematocrit (p<0.01) in comparison with controls. Group mean lymphocyte counts in these males were also slightly but statistically significantly lower than controls (p<0.05). The corresponding values in females previously given the high dose were similar to controls, however group mean neutrophil count in these females was statistically significantly higher than controls (p<0.05). The majority of individual values were within the background data ranges and these observations were considered unlikely to be of any toxicological significance.
CLINICAL CHEMISTRY
No toxicologically significant effects were detected in animals of either sex at any dose level.
At the end of the dosing period, males given test item at dose of levels of 1000 or 300 mg/kg bw/day showed statistically significantly higher values of bilirubin when compared with controls (p<0.05). The mean bilirubin value in males receiving the test item at 100 mg/kg bw/day was also slightly higher than controls, albeit without achieving statistical significance or showing clear dose-relationship. Group mean plasma concentration of glucose in females from the high dose group was also statistically significantly higher than controls (p<0.05) but without a dose relationship. The majority of individual values were within the background data ranges and the corresponding values from the recovery animals at the end of the treatment-free period were similar to controls. In the absence of any associated histopathology findings, these observations were considered unlikely to be of any toxicological significance. Any other intergroup differences achieving statistical significance were not dose-related and considered unrelated to treatment with the test item.
At the end of the treatment-free period, group mean albumin and alkaline phosphatase plasma concentrations in males previously given the high dose were statistically significantly higher than controls (p<0.05 and p<0.01, respectively). The corresponding values in females from this dose group were similar to controls and with all individual values from the males being within the background data ranges, these observations were considered unlikely to be of any toxicological significance.
URINALYSIS
No toxicologically significant effects were detected in males at any dose level.
At end of the treatment and treatment-free periods, urine samples from some treated males from all dose groups tested positive for the presence of protein. Since urine samples from one control non-recovery male and one control recovery male also tested positive for the presence of protein and there were no correlated histopathology observations, this finding was considered unlikely to be of any toxicological significance. The remaining urinalysis parameters appeared to be similar across all groups including controls.
NEUROBEHAVIOUR
Behavioral Assessments - No treatment-related changes in the behavioural parameters were detected at any dose level.
Functional Performance Tests - There was considered to be no effect of treatment with the test item at any dose level on functional performance in animals of either sex.
Any intergroup differences achieving statistical significance were not dose-related and considered to be unrelated to treatment with the test item.
Sensory Reactivity Assessments - Sensory reactivity scores across all test item-treated groups were similar to controls.
ORGAN WEIGHTS
At the end of the treatment period, males given the test item at a dose level of 1000 mg/kg bw/day showed statistically significantly higher absolute and body weight-related liver weights when compared with controls (p<0.05); the corresponding weights in high dose females were similar to controls. There was no dose-relationship, the majority of individual values from the treated males were within the background data ranges and there were no associated histopathology
abnormalities. This finding, was therefore, considered unlikely to be of any toxicological significance.
Group mean absolute and body weight-related thyroid/parathyroid weights in 1000 mg/kg bw/day females at the end of the treatment and treatment-free periods were statistically significantly lower than controls (p<0.01 and p<0.05, respectively). The majority of individual values were, however, within the background data ranges and in the absence of any histopathology correlates, this finding was considered unlikely to be of any toxicological significance. Any other intergroup differences in females achieving statistical significance were not dose-related and considered unrelated to treatment with the test item.
At the end of the recovery period, other intergroup differences achieving statistical significance in animals previously given the test item at 1000 mg/kg bw/day included higher absolute and body weight-related prostate and adrenal weights in males (p<0.05) and higher absolute and body weight-related spleen weights in females. The majority of individual values were within the background data ranges and these differences were considered to be of no toxicological significance.
GROSS PATHOLOGY
There were no macroscopic abnormalities considered to be related to treatment with the test item at any dose level.
Incidental abnormalities included reddened lungs (one female given 100 mg/kg bw/day and one recovery female previously given the high dose) and sloughing of the glandular region in the stomach (one female given 300 mg/kg bw/day).
HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of tissues from the control and high dose groups did not reveal any treatment-related findings. Any histopathological findings were considered to have occurred spontaneously or post-mortem. In addition to the routine evaluation of the testes, the seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No treatment-related abnormalities were noted.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Results of formulation analysis

Week No.

Nominal concentration (mg/mL)

Concentration found

(mg/mL)

% of nominal

1

0

ND

-

 

16.7

16.2

97

 

50

51.7

103

 

167

161

97

2

0

ND

-

 

16.7

18.8

112

 

50

56.1

112

 

167

143

86

3

0

ND

-

 

16.7

19.0

114

 

50

44

88

 

167

238

143

4

0

ND

-

 

16.7

19.2

115

 

50

52.8

106

 

167

164

98

5

0

ND

-

 

16.7

17.0

102

 

50

58.2

116

 

167

161

97

6

0

ND

-

 

16.7

22.2

133

 

50

50.3

101

 

167

160

96

Applicant's summary and conclusion

Conclusions:
The oral administration of the test item to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day was well tolerated. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day, based on the absence of toxicologically significant or treatment-related findings.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development), to assess the ability of the animals to recover from any toxicity following the withdrawal of treatment and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 or 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for forty-two consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study.

Pairing of non-recovery animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected non-recovery males from each dose group during the final week of treatment, and for five selected parental females from each dose group on Day 4 post partum. Urinalysis was performed on five non-recovery males per dose group during the final week of treatment and five non-recovery males and females from each dose group were selected for hematology and blood chemistry assessments prior to termination.

Adult non-recovery males were terminated on Days 43 or 44, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on Day 26 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Following forty-two days of treatment, recovery group animals were maintained without treatment for a further fourteen days. Urinalysis was performed on all recovery group males during the final week of the treatment period. In addition, hematological and blood chemical assessments were performed on all recovery group animals at the end of the treatment-free period. These animals were then subjected to a gross necropsy, but as there were no treatment-related histopathology findings, histopathological examinations of tissues from these animals was not performed.

Results

Mortality - There were no unscheduled deaths on the study.

Clinical Observations - Throughout the study, there were no clinical signs considered to be related to the toxicity of the test item.

Behavioral Assessment - There were no treatment-related changes in the behavioral parameters measured.

Functional Performance Tests - There was no effect of treatment with the test item at any dose level on functional performance

in animals of either sex.

Sensory Reactivity Assessments - Sensory reactivity scores across all treated groups were similar to controls.

Body Weight - Body weight development for animals of either sex remained unaffected throughout the treatment and recovery period.

Food Consumption - No adverse effects on dietary intake were noted for males during the study or for females during the pre-pairing, gestation or lactation phases of the study. Dietary intake and food efficiency in recovery animals of both sexes was also unaffected

.

Water Consumption - Visual inspection of water bottles did not indicate any differences in water intake for treated animals of either sex in comparison with controls.

Hematology - No toxicologically significant effects were detected in animals of either sex receiving the test item at any dose level.

Blood Chemistry - No toxicologically significant effects were detected in animals of either sex receiving the test item at any dose level.

Urinalysis - No toxicologically significant effects were detected in animals of either sex receiving the test item at any dose level.

Necropsy - No treatment-related macroscopic observations were detected at any dose level.

Organ Weights - At the end of the dosing period, group mean absolute and body weight-related liver weights in males given the test item at 1000 mg/kg bw/day were slightly but statistically significantly higher than controls. Group mean absolute and body weight-related thyroid weights in the females from this dose group were slightly but statistically significantly lower than controls at the end of the treatment and recovery periods. There were no histopathology correlates, and these observations along with any other statistically significant differences were considered to be of no toxicological relevance.

Histopathology - Microscopic examination of tissues from the control and high dose groups did not reveal any treatment-related findings.

Conclusion

The oral administration of the test item to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day was well tolerated. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day, based on the absence of toxicologically significant or treatment-related findings.