TROYSOL LAC

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-01-15 and 2008-04-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

27 July 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

30 September 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

liquid

Specific details on test material used for the study:

- Lac Technical with a purity of 98% (substance to registered without water)

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Recognized by international guidelines as a recommended rodent test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Laboratory Animal Services, Switzerland
- Weight at study initiation: 177 to 193 g (males), 139 to 157 g (females)
- Housing: in groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding
- Diet: ad libitum (Pelleted standard Kliba Nafag 3433)
- Water: ad libitum (tap water)
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: The feed batch and tap water was analyzed for contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a glass beaker on a tared Mettler balance. A small amount of vehicle was added, mixed and thereafter the remaining vehicle was added. The mixtures were stirred using a magnetic stirrer and stored at room temperature (15 -
25 °C).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

After experimental start, samples of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration and stability. Samples of about 2 g of each concentration were taken during week 3 after commencement of dosing to confirm homogeneity and concentration.The samples were delivered at ambient temperature to the Analytical Department and then stored at -20 ± 5 °C until analysis. The test item concentrations were determined by HPLC coupled to an UV detector and quantified with the area under the peak.
The identity of the test item was confirmed by its retention time which was similar to that measured in the working standards. The test item content in the samples, except six, was found to be within the accepted range of ± 20% of the nominal content. In addition, the homogenous distribution of the test item in PEG 300 was demonstrated. The application formulations were considered to be stable for at least 2 hours and 7 days when kept at room temperature.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats (RCC Study Number B63448), in which slight reductions of food consumption were noted initially in both sexes at 1000 and 600 mg/kg/day, and slightly reduced body weights were noted at 1000 and 600 mg/kg/day in both sexes and at 200 mg/kg/day in females. A slight reduction in the testes-to-body weight ratio was noted in males treated with 1000 mg/kg/day.

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed for clinical signs once before commencement of administration as well as twice daily on days 1 to 3, and once daily on days 4-28 (treatment period).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: These observations were performed once before commencement of administration and once weekly (weeks 1 to 3) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during acclimatization, treatment and recovery periods and before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule: once during the acclimatization period and weekly thereafter

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No. 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4
- Dose groups that were examined: in all animals
- Battery of functions tested: grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (adrenal glands, brain (including section of medulla/pons, cerebral and cerebellar cortex), bone marrow (femur), cecum, colon, duodenum, epididymides, heart incl. auricles, ileum with Peyer's patch, Jejunum with Peyer's patch, kidneys, liver, lungs, lymph nodes (mesentric and mandibular), ovaries, prostate gland incl. coagulating glands, rectum, sciatic nerve, seminal vesicles, spinal cord (cervical, midthoracic, lumbar), spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus, vagina and all gross lesions)

OTHER: Organ weights (brain, heart, liver, thymus, kidneys, adrenals, spleen, testes, epidymides, ovaries)

Statistics:

The following statistical methods used to analyze body weight, clinical laboratory data, grip strength and locomotor activity, organ weights and ratios as well as macroscopic findings:
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results".

Mortality:

no mortality observed

Description (incidence):

Please refer to "details on results".

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Please refer to "details on results".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Please refer to "details on results".

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Please refer to "details on results".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Please refer to "details on results".

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results".

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Please refer to "details on results".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Please refer to "details on results".

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Please refer to "details on results".

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

MORTALITY
All animals survived until scheduled necropsy.

CLINICAL SIGNS (DAILY AND WEEKLY)
During daily observations, soft feces were noted in rats at all dose levels. This finding is sometimes seen in studies using PEG 300 as vehicle. In the absence of similar changes in the control group, this was considered to be a possibly enhanced, non-adverse effect of the test item.
No changes or findings were noted during weekly detailed clinical observations (weeks 1-3).

FUNCTIONAL OBSERVATIONAL BATTERY
No changes or findings were noted during functional observational battery (week 4).

- Grip strength: The mean fore and hind limb grip strength values of the test item-treated males and females were considered to be unaffected by the treatment with the test item.

- Locomotor Activity: The mean locomotor activity of the test item-treated rats showed dose-unrelated and incidental differences when compared with the respective controls.

FOOD CONSUMPTION
At 1000, 300 and 100 mg/kg bw/day, the mean daily food consumption of test item-treated males and females was less than that of their respective controls during all measurement intervals.

BODY WEIGHTS
A test item-related reduction in mean absolute body weights and mean body weight gain were noted in males treated with 1000 mg/kg bw/day from day 8 of treatment onwards. Minor differences seen in the females at this dose level were considered to be insufficient to be a test item-related effect. The mean absolute body weights and mean body weight gain of the groups treated with 100
mg/kg bw/day or 300 mg/kg bw/day were generally similar to those of the controls.

HEMATOLOGY
The males and females treated with 1000 mg/kg bw/day had elevated white blood cell counts when compared with the respective controls, which resulted primarily from elevated lymphocyte counts in both sexes and in elevated neutrophils in males. These differences, although they remained within the ranges of the historical control values, were considered to be indicative of a mild inflammatory reaction. The remaining differences seen in the hematology parameters were considered to be unrelated to the treatment with the test item.

CLINICAL BIOCHEMISTRY
Liver-specific changes included elevations of aspartate aminotransferase activity in both sexes at 1000 mg/kg bw/day when compared with controls. Alanine aminotransferase activity was elevated in males at 300 mg/kg bw/day and in both sexes at 1000 mg/kg bw/day. The slight elevation in the glutamate dehydrogenase activity in both sexes at 1000 mg/kg bw/day was also considered to be an indication of hepatic involvement. These changes were considered to indicate elevated metabolism in the liver as a consequence of the test item. The slight reduction in blood glucose seen in rats treated with 1000 mg/kg bw/day may be related to the reductions in food consumption or a mild hepatic insufficiency.
Markedly elevated creatine kinase levels in rats treated with 1000 mg/kg bw/day were considered to be related to the test item as a possible secondary effect of muscle mass loss which culminated in the body weight changes. The elevated creatinine noted in rats at 1000 mg/kg/day was also considered to be related to this finding.
Elevated potassium levels were noted in males at 300 mg/kg bw/day and in both sexes at 1000 mg/kg bw/day; phosphorus levels were elevated in females at 1000 mg/kg bw/day.
Protein was reduced in both sexes at 1000 mg/kg bw/day, primarily as a result of reduced globulin, and with a concomitant increase in albumin/globulin ratios.

ORGAN WIGHTS
Test item-related changes in the mean absolute organ weights and/or ratios were noted at 1000 mg/kg bw/day in the heart, liver, thymus, adrenal and spleen. At 300 mg/kg bw/day, changes in liver weight and/or ratios were considered to be test item-related.

MACROSCOPIC/MICROSCOPIC FINDINGS
Test item-related macroscopical changes included crateriform retractions in the stomachs of both sexes at 1000 mg/kg bw/day. Liver enlargement was restricted to males treated with 300 mg/kg bw/day or 1000 mg/kg bw/day, but was considered to be test item related.

Microscopically, hepatocellular hypertrophy (adaptive in nature) was noted in the livers of males treated with 300 mg/kg bw/day and in both sexes treated with 1000 mg/kg bw/day.
In the stomach of females treated with 300 mg/kg bw/day, squamous cell hyperplasia and hyperkeratosis were noted. These changes were also seen in both sexes treated with 1000 mg/kg bw/day. Parakeratosis, submucosal inflammation, submucosal edema, and pustules were recorded in male and females treated with 1000 mg/kg bw/day, whereas forestomach ulceration and erosion were recorded in females. These findings were considered to be adverse.
Thymus atrophy was noted with increased incidence in both sexes treated with 1000 mg/kg bw/day, whereas the affected females also showed a minimal increase of severity grade of this finding, likely due to stress.
Atrophy of adrenal cortices was recorded in one female treated with 1000 mg/kg bw/day. The reason of this single case of adrenal atrophy remains unclear.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

1

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

2

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

3

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

immune system

Organ:

thymus

Treatment related:

yes

Dose response relationship:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, 100 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL). Based upon the microscopical changes in the stomachs of rats treated with 300 mg/kg/day or 1000 mg/kg/day, the no-observed-adverse-effect-level (NOAEL) was considered to be 100 mg/kg/day.

Executive summary:

In this sub-acute toxicity study, the test item was administered daily by oral gavage to Wistar rats of both sexes at dose levels of 100, 300 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, PEG 300, only.

The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest and the treatment periods. Functional observational battery, locomotor activity and grip strength were performed during week 4.

At the end of the dosing period, blood samples were withdrawn for hematology and plasma chemistry analyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, as well as liver, stomach, thymus of both sexes, adrenal glands of females of the mid- and low dose group, and all gross lesions from all animals.

No mortality, no changes during daily observations or weekly detailed observations (weeks 1-3), and no changes during the functional observational battery performed during week 4 (with no changes in the mean locomotor activity or the mean fore limb or hind limb grip strength values) were observed.

 

The mean daily food consumption of test item-treated males and females was less than that of their respective controls. Additionally, test item-related findings included reduced body weight development at 1000 mg/kg/day, minor changes in the hematology parameters (typical of a mild inflammatory reaction) at 1000 mg/kg/day, mild activation of liver enzymes (considered to be a metabolic reaction of adaptive nature), elevated potassium in males at 300 mg/kg/day and both sexes at 1000 mg/kg/day, increased phosphorus levels in females at 1000 mg/kg/day, macroscopically evident crateriform retractions in the stomachs of rats at 1000 mg/kg/day, and liver enlargement in males at 300 mg/kg/day and 1000 mg/kg/day, as well as reduced mean absolute organ weights and/or ratios in the heart, thymus, adrenal and spleen at 1000 mg/kg/day, increased liver weights and/or ratios at 1000 mg/kg/day and 300 mg/kg/day. Microscopically, squamous cell hyperplasia and hyperkeratosis were noted in the stomach of females treated with 300 mg/kg/day and in both sexes treated with 1000 mg/kg/day, parakeratosis, submucosal inflammation, submucosal edema, and pustules recorded in male and females treated with 1000 mg/kg/day, whereas forestomach ulceration and erosion were recorded in females. These findings were considered to be adverse.

 

Based on the results of this study, 100 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL). Based upon the microscopical changes in the stomachs of rats treated with 300 mg/kg/day or 1000 mg/kg/day, the no-observed adverse-effect-level (NOAEL) was considered to be 100 mg/kg/day.