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Toxicological information

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:
08-Fbruary-2013 to 19-March-2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed under GLP and based on OECD guideline no. 407 with some restrictions regarding the numbers of animals used in the study and organs examined.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
The number of animals usd was reduced and histopathology examination limited to the liver.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: compact
Details on test material:
-Identification: NPG-THPA hemiester (TK 12094).
-Molecular formula: C21H28O8.
-Molecular weight: 408.44
-CAS Number: 41026-17-9.
-Description: White to light yellow solid.
-Batch: Colkb03.
-Purity/Composition: 96%
-Test substance storage: At room temperature protected from light.
-Stability under storage conditions: Stable.
-Expiry date: 01 February 2014.
-Hygroscopic: Yes, store in well-sealed container.
-Volatile: No.
-Reactivity: Use amber-coloured glassware or wrap container in aluminium-foil.

Test animals

Species:
rat
Strain:
other: CrI:WI(Han) (Outbred, SPF-Quality)
Sex:
male/female
Details on test animals and environmental conditions:
Test System Rat: Crl:WI(Han) (outbred, SPF-Quality).
Rationale Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).
Source Charles River Deutschland, Sulzfeld, Germany.
Total number of animals 12 males and 12 females (females were nulliparous and non-pregnant).
Age at start of treatment Approximately 6 weeks.
Identification Earmark and tattoo.
Randomization By computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Acclimatization period At least 5 days before the start of treatment under laboratory conditions.
Health inspection Upon receipt of the animals.

Conditions
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation
Group housing of 3 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).

Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water
Free access to tap water

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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
Vehicle Propylene glycol (Merck, Darmstadt, Germany), specific gravity 1.036
Rationale for vehicle Based on trial formulations performed at WIL Research Europe and on information from the sponsor.
Method of formulation Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle and the density of the test substance. No correction was made for the purity of the test substance.
Storage conditions At ambient temperature.

Dose volume: 10 mL/kg body weight. Actual doses volumes were calculated weekly according to the latest body weight.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
150, 500 and 1000 mg/kg body weight
Basis:
actual ingested
No. of animals per sex per dose:
3 males and 3 females
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
Mortality / Viability At least twice daily.

Clinical signs Detailed clinical observations were made in all animals at least once during the following observation intervals:

Actual time after dosing Reported as hours after dosing Nominal time#
0-15 min 0 hour 7:00
1 hr ± 15 min 1 hour 8:00
3 hr ± 30 min 3 hours 10:00
# Nominal times were used for computer registration only

The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Body weights Weekly.

Food consumption Weekly.

Water consumption Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Blood samples were collected under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m. at the end of the treatment. Animals were deprived of food overnight (for a maximum of 24 hours), but water was available.





Sacrifice and pathology:
Animals surviving to the end of the observation period were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination.

The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy:

Adrenal glands, Spleen, Heart, Testes, Kidneys, Thymus, Liver.

The liver slides of all control animals and high dose animals were examined by a pathologist.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The clinical signs, such as rales, hunched posture and piloerection, were considered not toxicologically relevant based on the low incidence and low severity of the symptoms and the occurrence in also the control males.
Mortality:
mortality observed, treatment-related
Description (incidence):
The clinical signs, such as rales, hunched posture and piloerection, were considered not toxicologically relevant based on the low incidence and low severity of the symptoms and the occurrence in also the control males.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A higher liver weight in females was noted at 500 and 1000 mg/kg/day.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Males and females at 1000 mg/kg showed rales, hunched posture and/or piloerection during the study period. Rales were also noted at a lower incidence in males of control group and treated at 150 and 500 mg/kg.

Salivation, as seen after dosing among the animals of all groups, was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance.


BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.


HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats.

Minor dose related differences were noted in heamaglobin level, mean corpuscular heamoglobin, mean corpuscular heamoglobin concentration and reticulocytes counts in females. These changes were considered to be of no toxicological significance since the values were within the range considered normal for rats of this age and strain and no corroborative findings were noted.


CLINICAL CHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.

Values in treated females showing a trend over the dose groups were considered to have arisen as a result of slightly high or low control values and/or in the absence of a treatment-related distribution or corroborative findings in the opposite sex, considered to be of no toxicological significance.


ORGAN WEIGHTS
A higher relative liver weight in females was noted at 500 and 1000 mg/kg.

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations.

One control male showed foci in the thymus. This finding is occasionally seen among rats used in these types of studies. Since it is once seen in one control animal, it is considered not to be toxicologically significant.


HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings recorded in the liver which could be attributed to treatment with the test substance.

All microscopic findings in the liver were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.



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

Applicant's summary and conclusion

Conclusions:
Based on the absence of functional or morphological disturbances supporting the change in liver weight noted for NPG-THPA hemiester (TK 12094), a No Observed Adverse Effect Level (NOAEL) of 1000 mg/kg was established.
Executive summary:

A Repeated dose 28-day oral range finding study with NPG-THPA hemiester (TK 12094) by daily gavage in the rat was performed according to the following guidelines (the guidelines were not fully applicable as this study was mainly intended for dose selection): EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008   and OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008 .  

 

The dose levels for this 28-day toxicity study were selected to be 0, 500, 150 and 1000 mg/kg.

 

The test substance, formulated in propylene glycol, was administered daily for 28 days by oral gavage to-bred Wistar rats. One control group and three treated groups were tested, each consisting of 3 males and 3 females.

 

The following parameters were evaluated: clinical signs daily; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues. clinical pathology, macroscopy and organ weights at termination.

 

There were no changes at determination of body weight and food consumption measurements, or alterations during clinical laboratory investigations, macroscopic and microscopic examination that were considered to be an effect of treatment.

 

The clinical signs, such as rales, hunched posture and piloerection, were considered not toxicologically relevant based on the low incidence and low severity of the symptoms and the occurrence in also the control males. 

 

The slightly higher liver weights as noted in females at 500 and 1000 mg/kg were not accompanied by any related clinical biochemistry parameter or morphological changes. Therefore no toxicological significance was ascribed to this finding.