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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 15 Sep 2011 to 29 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 407, GLP-compliant)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Guidelines for Screening Toxicity Testing of Chemicals: Testing Methods for New Substances, enacted July 13, 1974, amended December 5, 1986.
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Source: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age (at Delivery): 7 weeks
- Body Weight Range (at Acclimatization): 192 to 207 g (mean 198 g) in males and 122 to 150 g (mean 137 g) in females
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey, UK)
- Diet: Pelleted standard Harlan Teklad 2914C (batch nos. 44/11 and 46/11) rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batches were analyzed for contaminants.
- Water: Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: yes, 6 days
- Randomization: Randomly allocated to groups by body weight.

ENVIRONMENTAL CONDITIONS
Standard laboratory conditions, continuously monitored.
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 to 70
- Air changes (per hr): . Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (with at least eight hours music during the light period)

IN-LIFE DATES: From: 22 SEP 2011 To: 20 OCT 2011 (recovery groups of control and high dose group were necropsied at 3rd NOV 2011)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Method:
- Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
- Frequency of Administration: Daily.
- Dose Levels:
Group 1: 0 mg/kg/day
Group 2: 100 mg/kg/day
Group 3: 300 mg/kg/day
Group 4: 1000 mg/kg/day
- Rationale for Dose Level Selection:The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats, Harlan Laboratories study.
- Dose Volume: 10 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The application formulations investigated during the study were found to comprise test item in the range of 87.1% to 105.3%, and met the required content limit of ±20% with reference to the nominal content. Because single results found did not deviate more than 10% (<15%) from the corresponding mean, dose formulations with test item were considered to be homogeneous.
The test item was found to be stable in application formulations of group 4 (concentration of 100 mg/mL) when kept four hours or eight days under room temperature: recoveries met the variation limit of 10% from the time-zero (homogeneity) mean.
However, the result of stability in groups 2 and 3 exceeded the acceptance criteria. In application formulations of group 2 (10 mg/mL), the maximum deviation of time-zero mean was found to be 24.0% for 4 hours stability and 23.4% for eight day stability, as in application formulations of group 3 (30 mg/mL), the maximum deviation of time-zero mean was found to be 14.8% for 4 hours stability and 16.1% for eight day stability.
Although it is not possible to determine the exact reason for exceeding the acceptance criteria, smaller dose levels commonly yield greater deviation from time zero after 4 hours and changes in the recovery do not change between sampling intervals (4 hours and 8 days). A possible explanation is an undetermined degree of reactivity with a constant amount of another substance such as oxygen. It is also possible that adsorption to the surface of the sample vessel may have occurred. In addition, a lack of stability does not stop after 4 hours.
In conclusion, the results indicate the accurate use of the test item and bidistilled water as vehicle during this study. Application formulations were found to be homogeneously prepared.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0 , 100, 300, 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
Group 1 (0 mg/kg/day): 10 males / 10 females
Group 2 (100 mg/kg/day): 5 males / 5 females
Group 3 (300 mg/kg/day): 5 males / 5 females
Group 4 (1000 mg/kg/day): 10 males / 10 females
Control animals:
yes
Details on study design:
The purpose of this oral toxicity study was to assess the cumulative toxicity of the test item when administered daily to rats by gavage for a period of 28 days. The reversibility of treatment-related changes was assessed after a treatment-free 14-day recovery period.

Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (for viability/mortality)

CLINICAL OBSERVATIONS: Yes
- Time schedule: daily (during acclimatisation period, before test item administration during treatment period and during recovery period; twice daily during days 1 to 3 of treatment period)

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes ( for details see "any othe information on materials and methods")
- Time schedule for collection of blood: At the end of the dosing and the treatment-free recovery period

CLINICAL CHEMISTRY: Yes ( for details see "any othe information on materials and methods)
- Time schedule for collection of blood: At the end of the dosing and the treatment-free recovery period

URINALYSIS: Yes ( for details see "any othe information on materials and methods")

NEUROBEHAVIOURAL EXAMINATION: Yes ( for details see "any othe information on materials and methods")
- Time schedule for examinations:during week 4
- Battery of functions tested: grip strength / locomotor activity

OTHER:Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. The stage of estrus was also determined by the pathologist.
Sacrifice and pathology:
GROSS PATHOLOGY:Yes ( for details see "any othe information on materials and methods")
HISTOPATHOLOGY: Yes (performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals)
A description of all abnormalities is included. Attempts were made to correlate gross observations with microscopic findings.
Statistics:
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:
no effects observed
Description (incidence and severity):
one test item-unrelated mortality
Mortality:
no mortality observed
Description (incidence):
one test item-unrelated mortality
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:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Not test item related, considered to be secondary findings after aspiration
Histopathological findings: neoplastic:
not examined
Details on results:
Observations
Viability / Mortality
All males and females survived the treatment period. On day 1 of the recovery period, female no. 58 (previously treated with 1000 mg/kg/day) died during anesthesia for blood sampling.

Daily Observations
No test item-related adverse findings were noted during the treatment period and no late effects were noted during the recovery period. At all dose levels, yellow staining of the feces was noted with dose-dependent onset; this was considered to be a passive effect and is commonly noted following oral administration of a dyestuff. In males and females at 100 mg/kg/day, this was noted on day 25; at 300 mg/kg/day on day 7 and from day 25-28 and at 1000 mg/kg/day from day 7 onwards.
Minor scabbing was noted on the shoulders of one male treated with 1000 mg/kg/day on days 26-28 of treatment and in one female treated with 300 mg/kg/day on day 28 of treatment. These findings were considered to be unrelated to the test item.
During the recovery period, fecal staining was reversible from day 3 onwards.

Weekly Behavioral Observations
No findings were evident during the weekly behavioral observations performed at weeks 1, 2 or 3 of treatment.

Functional Observational Battery (Screen)
No findings were evident during the functional observational battery performed at week 4 of treatment.

Grip Strength
The mean fore- and hind limb grip strength values of the test item-treated males and females compared favorably with those of the respective control values.

Locomotor Activity
The mean locomotor activity values of the test item-treated males and females were considered to be unaffected. During the first measurement interval (0-10 minutes), significantly higher values were noted in females (p<0.05) treated with 1000 mg/kg/day when compared with the controls. All other values recorded during subsequent measurement intervals were similar to those of the control females.

Food Consumption
There were no test item-related changes in the mean daily food consumption at any dose level.
The mean daily food consumption of the test item-treated females decreased slightly during the recovery period, but this was considered to be an artifact caused by the coincidental removal (and necropsy) of the cage with the higher mean food consumption.

Body Weights
The mean absolute and relative body weights of the test item-treated rats compared favorably with their respective control values during the treatment period. During the recovery period, significantly lower (p<0.01) mean body weight gain values were noted in the males (days 8 and 14) and females (day 8) previously treated with 1000 mg/kg/day.

Clinical Laboratory Investigations
Hematology
There were no test item-related effects upon the hematology parameters of males or females at any dose level.
At 1000 mg/kg/day, significantly elevated mean corpuscular volume (p<0.05) was noted at the end of the treatment period in males when compared with the controls. This difference remained within the range of the historical control data and was considered to be incidental.
The mean absolute and relative reticulocyte counts of the males treated with 1000 mg/kg/day were significantly elevated when compared with the controls. However, when compared with the historical control data, the control values were lower and therefore the increased values were considered to be an artifact.
All other changes of statistical significance were noted in the low- or middle-dose groups and were without dose dependence. Therefore these differences were considered to be unrelated to the test item.
After the recovery period, the hematology parameters of the males compared favorably. In females previously treated with 1000 mg/kg/day, significantly lower mean corpuscular volume (p<0.05), the mean absolute eosinophil count (p<0.01) and mean absolute basophil count (p<0.05) were noted when compared with the respective control values, but all values remained within the ranges of the historical control values.

Clinical Biochemistry
There were no test item-related effects upon the clinical biochemistry parameters of males or females at any dose level.
After the treatment period, the mean sodium level was significantly elevated in males treated with 1000 mg/kg/day (p<0.05) when compared with the controls and marginally exceeded the upper range of the historical control data. No toxicological relevance was associated with this isolated difference.
All clinical biochemistry values recorded in the females treated with 1000 mg/kg/day were similar to those of the control females. All other changes of statistical significance were noted in the low- or middle-dose groups and were without dose dependence. Therefore these differences were considered to be unrelated to the test item.
After the recovery period, males which were previously treated with the test item at 1000 mg/kg/day showed significantly elevated glucose levels (p<0.05) and elevated triglycerides (p<0.01) which remained within the ranges of the respective historical control values. The mean phospholipid level was significantly elevated (p<0.01) and exceeded the upper limit of the historical control data, but in the absence of similar difference after the end of the treatment period, was considered to be of no toxicological relevance. In females, the mean phospholipid level was significantly reduced (p<0.05) when compared with control females. Significant reductions of creatine kinase (p<0.05), calcium (p<0.01), phosphorus (p<0.01) protein (p<0.05) and albumin (p<0.05) were noted, all of which remained within the range of the historical control data.

Urinalysis
There were no test item-related effects upon the urinalysis parameters of males or females at any dose level.

Pathology
Organ Weights
At the end of the treatment period, there were no statistically significant differences in the mean absolute and relative organ weights at any dose level. During the recovery period, the mean absolute adrenal weights and mean absolute uterus weights (including oviducts) were significantly elevated (p<0.05) in females previously treated with 1000 mg/kg/day. The mean heart-to-body weight ratio of these females was marginally, but significantly, reduced (p<0.05), whereas significantly higher organ-to-brain weights were noted for pituitary (p<0.05), thyroids (p<0.05), adrenals (p<0.05) and uterus/oviducts (p<0.01) when compared with the controls. Insofar as these findings were not evident at the end of the treatment period and were not accompanied by microscopical changes of morphology, all were considered to be unrelated to the treatment with the test item.

Macroscopic Findings
After the treatment period, a small number of macroscopical changes were noted in rats at all dose levels, with no clear dose dependent incidence.
In males, discoloration and/or inflated lungs were noted in single males at 100 and 1000 mg/kg/day. Inflated lungs were also recorded in two females at 100 mg/kg/day. Lung foci were recorded for one female at 1000 mg/kg/day.
Thymic foci and/or discoloration were noted in on control female, one female at 100 mg/kg/day, two females at 300 mg/kg/day and one male at 1000 mg/kg/day.
Enlargement and/or discoloration of the lymph nodes were noted in one male treated with 100 mg/kg/day and in one male treated with 1000 mg/kg/day.
Uterine dilation was recorded in one control female and single females at 300 mg/kg/day and 1000 mg/kg/day. Skin sores were recorded in one female at 300 mg/kg/day and in one male at 1000 mg/kg/day,
After the recovery period, one male previously treated with 1000 mg/kg/day had smaller testes and epididymides and a female had discoloration of the lungs and ovaries. These findings were not considered to be related to late effects of the test item.

Microscopic Findings
Lung, Trachea
In male number 14 (100 mg/kg/day) and in female number 53 (1000 mg/kg/day), fine granular yellowish foreign material was found to be present in alveolar macrophages. Similar foreign material was found in inflammatory (granulomatous) lesions of the lung of male number 25 (1000 mg/kg/day) and in the submucosal areas of the trachea of this animal.

Lymph Nodes
The above animals showed macroscopically enlarged and yellowish discolored bronchial and mediastinal lymph nodes. During microscopic examination lymphoid hyperplasia along with accumulated fine granular, yellowish foreign material was recorded.

Other Findings
The remaining findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant findings were noted up to the dose level of 1000 mg/kg bw/day, the highest dose level tested.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this OECD 407 subacute study, a no-observed-effect-level (NOEL) could not be established but the no-observed-adverse-effect-level (NOAEL) was considered to be 1000 mg/kg body weight/day.
Executive summary:

In this subacute toxicity study, the test material 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, bidistilled water, only. The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during acclimatization, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

There were no toxicologically relevant deaths. One high dose female died during anesthesia for blood sampling.

No test item-related adverse findings were noted in the daily clinical observations performed during the treatment period and no late effects were noted during the recovery period. At all dose levels, yellow staining of the feces was noted with dose-dependent onset; this was considered to

be a passive effect and is commonly noted following oral administration of a dyestuff. No findings were evident during the weekly behavioral observations performed at weeks 1, 2 or 3 of treatment.

No findings were evident during the functional observational battery performed at week 4 of treatment.

The mean fore- and hind limb grip strength values of the test item-treated males and females compared favorably with those of the respective control values.

The mean locomotor activity values of the test item-treated males and females were considered to be unaffected.

There were no test item-related changes in the mean daily food consumption at any dose level.

The mean absolute and relative body weights of the test item-treated rats compared favorably with their respective control values during the treatment period. Minor differences seen during the recovery period were considered to be of no toxicological relevance.

There were no test item-related effects upon the hematology paramters of males or females at any dose level.

There were no test item-related effects upon the clinical biochemistry parameters of males or females at any dose level.

There were no test item-related effects upon the urinalysis parameters of males or females at any dose level.

At the end of the treatment period, there were no statistically significant differences in the mean absolute and relative organ weights at any dose level. During the recovery period, the mean absolute adrenal weights and mean absolute uterus weights (including oviducts) were elevated in females previously treated with 1000 mg/kg/day. The mean heart-to-body weight ratio of these females was marginally reduced, whereas higher organ-tobrain weights were noted for pituitary, thyroids, adrenals and uterus/oviducts when compared with the controls. Insofar as these findings were not evident at the end of the treatment period and were not accompanied by microscopical changes of morphology, all were considered to be

unrelated to the treatment with the test item.

After the treatment period, a small number of macroscopical changes were noted in rats at all dose levels, with no clear dose dependent incidence.

Microscopical changes were noted in the in lungs, trachea, bronchial and mediastinal lymph nodes of two single animals, and were considered to be largely secondary changes that were not indicative of systemic toxicity.

The only test item-related, yet non-adverse findings were restricted to staining of the feces in rats at all dose levels, which was considered to be a typical passive effect following large oral doses of a dyestuff.

Based on the results of this study, a no-observed-effect-level (NOEL) could no be established but the no-observed-adverse-effect-level (NOAEL) was considered to be 1000 mg/kg body weight/day.