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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 January 2017 to 06 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Purity: >90%
- Description: Brown viscous liquid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Housing
Animals were housed in a limited access room in cages comprised of a polycarbonate body with a stainless steel mesh lid. Solid polycarbonate bottom cages were used during the acclimatization, pre-pairing, treatment, gestation, littering and lactation periods. Solid bottom cages contained softwood based bark-free fibre bedding, which was changed at appropriate intervals each week. Grid bottomed polypropylene cages that were suspended above absorbent paper, which was changed daily, were used during pairing. The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.

Temperature and relative humidity were monitored and maintained within the range of 20-24ºC and 40-70%. The air supply was filtered fresh air, which was not recirculated. Artificial lighting was set to provide 12 hours light and 12 hours dark.

Number of animals per cage
Pre-pairing up to five animals of one sex
Pairing one male and one female
Males after mating up to five animals
Gestation one female
Lactation one female + litter

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
The Crl:CD(SD) rat strain was used because it is recognized by regulatory agencies as an appropriate model for conditions of possible human exposure and because of the historical control data available at the testing laboratory. The oral gavage route of administration, using a suitably graduated syringe and a rubber catheter inserted via the mouth, at doses of 0, 100, 330 and 1000 mg/kg/day in arachis oil BP, with a dose volume of 5 ml/kg, was chosen based upon results of a 14-day dose range finding oral gavagel toxicity study in the Crl:CD(SD) rat in which there were no mortalities and few signs of toxicity at the high dose of 1000 mg/kg bw.

A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mean concentrations of the test item in dose test formulations analyzed for the study were within ±12% of nominal concentrations, confirming accuracy of the dose formulations.
Duration of treatment / exposure:
Males: Two weeks before pairing up to necropsy after minimum of five weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
330 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The dose selection was based upon a range-finding study in which there were no mortalities ad few signs of toxicity at the high dose of 1000 mg/kg/bw (please see RSS section 7.5.1 Supporting study, Envigo, 2018 (range-finding).

Examinations

Observations and examinations performed and frequency:
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
F0 males Week 1 - daily
Week 2 onwards - once each week
F0 females Week 1 - daily
Week 2 - once
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12

Detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation
One to two hours after completion of dosing
As late as possible in the working day
Sacrifice and pathology:
All adult animals: Carbon dioxide asphyxiation.

Offspring
- selected for thyroid hormone sampling on Day 4 and 13 of age: Decapitation.
- all other: Intraperitoneal injection of sodium pentobarbitone.

Sequence: To allow satisfactory inter-group comparison.

All adult animals were subject to a detailed necropsy.
Other examinations:
A viability check was performed near the start and end of each working day. Animals were inspected at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Detailed physical examination and arena observations
Before treatment commenced, during each week of treatment, on Days 0, 7, 14 and 20 after mating, and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.

After removal from the home cage, animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behaviour. Findings were either reported as "present" or assigned a severity grade.

Sensory reactivity and grip strength
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Day 7-9 of lactation. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. The following measurements, reflexes and responses were recorded: approach response, pinna reflex, auditory startle reflex, tail pinch, and grip strength.

Motor activity
Motor activity of the five lowest numbered surviving males and the first five lactating females in each group was measured (before dosing) during Week 5 of treatment for males and at Day 7-9 of lactation for females.

Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity.

Body weight
The weight of animals was recorded as follows:
F0 males:
Weekly during acclimatization.
Before dosing on the day that treatment commenced (Day 1) and weekly thereafter.
On the day of necropsy.
F0 females:
Weekly during acclimatization.
Before dosing on Day 1 and weekly before pairing.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7 and 13 of lactation.
On the day of necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals Weekly, from the day that treatment commenced.
Food consumption was not recorded for males and females
during the period when paired for mating (Week 3), but
recommenced for males in Week 4.
For females after mating food consumption was performed to
match the body weight recording:
Days 0-6, 7-13 and 14-19 after mating
Days 1-3, 4-6 and 7-12 of lactation.
From these records, the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

Oestrous Cycles
Dry and wet smears were taken as follows:
Dry smears For 15 days before pairing using cotton swabs.
Wet smears Using pipette lavage during the following phases:
For 14 days before treatment (all females including
spares); animals that failed to exhibit 4-5 day cycles
were not allocated to study.
After pairing until mating.
For four days before scheduled termination (nominally Days 11 to 14 of lactation).
Statistics:
Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant. For some parameters, including estrous cycles before treatment, stage of estrous cycle at termination and gestation index the similarity of the data was such that analyses were not considered to be necessary. Statistical analyses were carried out separately for males and females using the individual animal as the experimental unit for litter/foetal findings, the litter was taken as the treated unit.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
One non-pregnant, non-littering female receiving 330 mg/kg/day was sacrificed on gestation day 25, as specified in the study plan.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
For females treated at 1000 mg/kg/day, mean food consumption was slightly high when compared with controls prior to pairing (Weeks 1-2) but these variations were marginal and considered incidental.

During gestation, food consumption was comparable to controls in females treated at 100, 330 or 1000 mg/kg/day.

During Days 4-7 and 7 -13 of lactation, mean food consumption was slightly high in females treated at 330 or 1000 mg/kg/day when compared with controls but these variations were marginal with no statistical significance and considered incidental.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological evaluation in males of the 330 or 1000 mg/kg/day groups revealed statistically significant decreases of mean prothrombin time (PT) and activated partial prothrombin time (APTT) compared to the concurrent control group.

In addition, for males of the 1000 mg/kg/day group, mean large unstained cells (LUC) and platelet counts were marginally higher than the concurrent control group. These differences were within the historical control data ranges and considered normal biological variations, not treatment related.

Hematological evaluation in females revealed no treatment-related effects.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Potassium concentration in males treated at 100, 330 or 1000 mg/kg/day were slightly high when compared with controls and phosphorus concentration in males and females treated at 100, 330 or 1000 mg/kg/day were also slightly high however these differences were slight and there was no evidence of a dose response.

Furthermore, these changes were within the historical control data ranges and are considered incidental.

Triglycerides were low in females treated at 100, 330 or 1000 mg/kg/day however, these values were within the historical control data ranges and considered incidental.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Sensory reactivity and grip strength were unaffected by treatment.

Group mean high and low beam activity scores for all groups of treated males were low compared with Controls, with statistical significance attained at the 12-minute interval (100 mg/kg/day; high and low beams), the 42-minute interval (1000 mg/kg/day; low beam only) and the total scores (all treated groups, high beams only). However, the majority of these scores were within the historical control data ranges, including some of the scores that have attained statistical significance.

The majority of group mean high and low beam activity scores for all groups of treated females were slightly high compared with Controls with an isolated statistical significance attained at the 42-minute interval for low beams but the differences in the total scores did not attain statistical significance and no effect of treatment is inferred.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean absolute and adjusted organ weights in males were unaffected by treatment.

Group mean adjusted heart weights were marginally high in females treated at 100, 330 or 1000 mg/kg/day attaining statistical significance at all dose levels however these weights lacked dose relationship and were within the historical control data ranges as such are not considered to be attributable to treatment.

Decreases in thymus weights were observed in females treated at 100, 330 or 1000 mg/kg/day with the difference from controls demonstrating a dose-relationship; these weights did not attain statistical significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopic examination performed in males after 5 weeks of treatment or in females on Day 14 of lactation revealed no test item-related lesions.

A kidney mass was observed on one male given 1000 mg/kg/day however this was an isolated incidence and was considered to be unrelated to treatment.

Pale areas were seen in the kidneys of several females, including in control animals; there was no relationship to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic examination revealed accumulations of macrophages which were present in the mesenteric lymph nodes of males and females of animals given 330 or 1000 mg/kg/day.

Vacuolation was present in the adrenal cortex (zona reticularis and zona fasciculata) of two males given 1000 mg/kg/day; as this was only present in two males and the severity of this finding was slight this finding could not be directly attributed to treatment.

A single incidence of nephroblastoma was observed in a male given 1000 mg/kg/day. These tumours do occasionally occur as spontaneous lesions in younger animals such as this in short term studies (Ikezaki et al, 2011). Given the short term nature of this study and the absence of similar lesions in other animals, it is considered that this is an incidental finding and not related to administration of the test item.

Mineralisation was present in the tubules of the renal cortex in females from all groups, including controls. Similar renal changes were not identified in males. The finding is not considered to be related to treatment.

Foci of mucosal congestion and epithelial necrosis were present in the glandular region of the stomach in females from all groups, including controls. These changes are not considered to be related to treatment. The cause of the lesions is unclear.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The No-observed-adverse-effect-level (NOAEL) of the test item for systemic toxicity effects was considered to be 1000 mg/kg/day.
Executive summary:

The study has been designed to assess the general systemic toxic potential in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item by oral gavage administration for at least five weeks. In addition, the study has been performed to the standardized guideline OECD 422, under GLP conditions.

 

Three groups of ten male and ten female rats received the test item at doses of 100, 330 or 1000 mg/kg/day in arachis oil by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, arachis oil, at the same volume dose as the treated groups.

 

During the study the following observations were made for the adult animals: clinical condition, sensory reactivity, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, serum thyroxine (T4) analysis, organ weight and macroscopic pathology and histopathology investigations were undertaken.

 

There were no treatment related deaths and no adverse effects on clinical condition, sensory reactivity, grip strength, motor activity, body weight gain, food intake, haematology, and blood chemistry measurements in males and females. 

 

There was no treatment related effect on the serum T4 levels in all analysed samples from adult male rats.

 

In conclusion, oral administration of the test item to parental Sprague Dawley (Crl:CD(SD)) rats at dose levels of 100, 330 or 1000 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 13 of lactation in females was well-tolerated with no adverse effect observed.

 

The No-observed-adverse-effect-level (NOAEL) of the test item for systemic toxicity effects was considered to be 1000 mg/kg/day.