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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:
05-Aug-2009 to 16-Feb-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in a GLP laboratory in compliance with agreed protocols, with no significant deviations from standard test guidelines or methodological deficiencies.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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
GLP compliance:
yes
Remarks:
Date of most recent GLP inspection: April 2010
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Animals:
Rat, RccHan: WIST(SPF)
Rationale:
Recognized by international guidelines as a recommended test system.
Breeder:
Harlan Laboratories B.V.
Kreuzelweg 53
5961 NM Horst / Netherlands
Number of Animals:
Group 1: 5 males and 5 females
Group 2: 5 males and 5 females
Group 3: 5 males and 5 females
Group 4: 5 males and 5 females
Reserve animals:
Group 10: 1 male and 1 female
Total Number of Animals Ordered:
21 males and 21 females
Age (at Delivery):
Ca. 7 weeks
Body Weight Range (at Acclimatization):
Males: 193 to 220 g (mean 204 g)
Females: 140 to 164 g (mean 152 g)
Identification:
Acclimatization: Cage card and tail mark (later ear tattoo)
Treatment: Cage card and individual ear tattoo

Acclimatization:
Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Conditions:
Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). There was 12-hour fluorescent light/12-hour dark cycle with music during the light period.
Accommodation:
In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland).
Diet:
Pelleted standard Kliba Nafag 3433 (batch no. 35/09) rat / mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.
Water:
Community tap-water from Itingen was available ad libitum in water bottles.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
Bidistilled water
Method of administration:
Gavage
Preparation of dosing solutions:
The dose formulations were prepared weekly. Based upon the results of dose formulation analyses performed during a non-GLP dose range finding study (Harlan Laboratories Study C46062), the stability of the test item formulations was considered to be sufficient to justify weekly preparation.
TRIQUAT MONOMER was weighed into a glass beaker on a tared Mettler balance. A small amount of vehicle was added, the mixture was stirred and then the remning vehicle was added. The mixtures were stirred using a magnetic stirrer and used at room temperature (20 ± 5 °C).
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Dose Volume:
10 mL/kg body weight
Dose Concentrations:
Group 1: 0 mg/mL/day
Group 2: 10 mg/mL/day
Group 3: 30 mg/mL/day
Group 4: 100 mg/mL/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analysis was performed by Harlan Laboratories Ltd. using a HPLC method provided by the Sponsor.
After experimental start and during week 3, duplicate 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. Duplicate samples of about 2 g of each concentration were taken to confirm stability (2 hours and 7 days). The samples were delivered to the Analytical Department (Harlan Laboratories Ltd., Analytics,
Itingen Switzerland) and stored there at -20 ± 5 °C until analysis.
The test item was used as analytical standard.
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime: 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
Actual dose levels (i.e. excluding the water content) of 100, 300 and 1000 mg/kg body weight/day
Basis:
actual ingested
No. of animals per sex per dose:
Male and Female: 5 animals per sex at 0 mg/kg/day
Male and Female: 5 animals per sex at 100 mg/kg/day
Male and Female: 5 animals per sex at 300 mg/kg/day
Male and Female: 5 animals per sex at 1000 mg/kg/day
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, Harlan Laboratories Study C46062.
- Rationale for animal assignment (if not random):
Random, stratified by body weight.
- Rationale for selecting satellite groups:
Not applicable
- Post-exposure recovery period in satellite groups:
Not applicable
Positive control:
None

Examinations

Observations and examinations performed and frequency:
MORTALITY:
Observations for viability / mortality were recorded twice daily.
CAGE SIDE OBSERVATIONS:
The animals were observed for clinical signs once before commencement of administration as well as daily on days 1 - 28 (twice daily during days 1 - 3) during the treatment period.
DETAILED CLINICAL OBSERVATIONS:
The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 3) thereafter.
BODY WEIGHT:
Individual body weights were recorded on Day 1 (prior to start of treatment) and on Days 8, 15, 22 and 28. Terminal bodyweights were also recorded at necropsy.
FOOD CONSUMPTION:
The food consumption was recorded once during the acclimatization period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.
WATER CONSUMPTION:
Not applicable
OPHTHALMOSCOPIC EXAMINATION: No data:
Not applicable
CLINICAL LABORATORY INVESTIGATIONS
- At the end of the treatment period (Day 28).
- Anesthetic used for blood collection: isoflurane
- Animals fasted:
Yes
- How many animals:
All
HEMATOLOGY: Yes
Routine hematological parameters were examined.
CLINICAL BIOCHEMISTRY: Yes
Routine biochemical parameters were examined.
URINALYSIS: No data
Not applicable
NEUROBEHAVIORAL EXAMINATION: Yes
- Time schedule for examinations: Week 4
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity.
Behavioral Assessments were undertaken for all animals at weekly intervals throughout the study. Motor Activity, Forelimb/Hindlimb Grip Strength and Sensory Reactivity were undertaken for all animals during the final week of treatment.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.

All animals were subjected to a full external and internal macroscopic examination and any abnormalities were recorded.

HISTOPATHOLOGY: Yes (see TABLES OF WEIGHED, FIXED AND EVALUATED ORGANS below )

All control and high dose animals were subjected to a full histological examination and low and intermediate group animals were routinely subjected to examination of liver and spleen.
Other examinations:
The organs were weighed before fixation and recorded on the scheduled dates of necropsy. Relative organ weights were calculated on the basis of the body weight and brain weight.
The terminal body weight was recorded immediately prior to necropsy and the organ to terminal body weight ratios as well as organ to brain weight ratios were determined.
Statistics:
All data was summarised in tabular form.
The following statistical methods were used to analyze body weight, grip strength, locomotor activity, clinical laboratory data, 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:
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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
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:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Observations
Viability / Mortality:
All animals survived the scheduled treatment period.

General Cageside Observations (Daily)
No test item-related changes were noted during the general cage side observations. Kinked tail was noted in a single male treated with 100 mg/kg/day. All other males were without clinical signs and there were no clinical signs noted in the females at any dose level.

Detailed Clinical Observations (Weekly)
No test item-related changes were noted in the detailed behavioral observations (weeks 1 - 3) at any dose level.

Functional Observational Battery
No test item-related changes were noted in the functional observational battery (week 4) at any dose level.
Grip Strength
No test item-related changes in the mean fore- and hindlimb grip strength were noted in males at any dose level.
In females treated with 1000 mg/kg/day, the mean fore limb grip strength values were significantly elevated (p<0.05) when compared with the control females. No differences were seen at lower dose levels and because the mean hind-limb grip strength values were unaffected in the females at 1000 mg/kg/day, the significant difference in the fore limb grip strength was considered to be incidental.

Locomotor Activity
No test item-related differences were noted in the mean locomotor activity of males at any time point.
In females treated with 1000 mg/kg/day, significantly increased locomotor activity was noted at 0-10 minutes (p<0.05), 10-20 minutes (p<0.01) and 20-30 minutes (p<0.05) when compared with the controls. Females treated with 300 mg/kg/day had significantly increased locomotor activity during 10-20 minutes (p<0.05) when compared with the respective controls.
Although possibly related to the administration of the test item, these differences were reversible within the 60-minute observation period and were absent in the males at these dose levels. Therefore, the differences were not considered to be adverse.

Food Consumption
The mean daily food consumption of the test-item treated males was similar to that of the control males. In females treated with 1000 mg/kg/day, slightly lower mean daily food consumption was considered to be not related to the treatment of the test item, since slightly lower values were already noted during the acclimatization period.

Body Weights
The mean body weights of the males and females treated with 1000 mg/kg/day were marginally lower than those of the respective controls, but the differences did not attain statistical significance and were generally similar to initial differences seen at the beginning of the treatment period. The mean body weight gain values were also marginally lower that those of the respective controls. These differences were considered to be unrelated to the test item.
At 300 mg/kg/day and 100 mg/kg/day, the mean body weights and the mean body weight gain of the test item-treated males and females compared favorably with that of the respective control values.

Clinical Laboratory Investigations
Hematology
There were no differences of statistical or toxicological relevance in the hematology parameters of any test item-treated male.
In females treated with 100 mg/kg/day or 1000 mg/kg/day, significantly reduced mean absolute basophil counts (p<0.05 and p<0.01, respectively) were noted when compared with the controls. In females treated with 300 mg/kg/day, a significantly reduced mean neutrophil count was noted when compared with controls. These differences were either not clearly dose related or remained within the ranges of the historical control data, and therefore considered to be incidental.

Clinical Biochemistry
None of the statistically significant changes seen in test item-treated rats were considered to be of toxicological relevance.
Although significantly lower globulin levels were noted in females at 100 mg/kg/day (p<0.05), 300 mg/kg/day (p<0.05) and 1000 mg/kg/day (p<0.01), these differences were considered to be an artifact and due to an outlying control value rather than to any changes of toxicological relevance. Tthe control values also exceeded the ranges of the historical control data.
At 1000 mg/kg/day, no differences of toxicological relevance were noted when compared with control values.
At 300 mg/kg/day, significantly elevated sodium was noted in males (p<0.05) when compared with controls. Similar differences were not seen in males at 1000 mg/kg/day and this was therefore considered to be incidental. Significantly reduced urea was noted in females when compared with controls.
At 100 mg/kg/day, males showed significantly elevated mean phospholipids (p<0.05) which was not seen at higher doses and therefore considered to be incidental. Significantly reduced urea was noted in females when compared with controls.

Estrus Stages
The frequency and duration of the estrous cycles were similar in the females of the control and test item-treated groups.

Pathology
Organ Weights
In test item-treated males, no statistically significant differences in the mean absolute or relative organ weights were noted when compared with the control males.
In females treated with 1000 mg/kg/day, the mean absolute liver weights were significantly reduced (p<0.05) when compared with the controls. The mean absolute and relative organ weights of all other treated females in all other groups were considered to be unaffected.

Macroscopic Findings
No macroscopical findings were evident in any female.
At 1000 mg/kg/day, one male (no. 17) showed reduced testes and epididymide sizes, and reddish foci on the thymus. A second male (no. 19) showed dark red foci on the mandibular lymph nodes.
At 100 mg/kg/day, one male (no. 7) showed a red focus on the lung, and a second male (no. 8) had a kinked tail.
These findings were considered to be unrelated to the treatment with the test item.

Microscopic Findings
All lesions recorded during the microscopic investigation were within the range of background alterations that may be recorded in this type of study, and in rats of this strain and age.

The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. All calibration points used met the acceptance limit of ±20% variation from the calibration curve derived by
linear regression analysis. The regression coefficient calculated was found to exceed 0.99. The TRIQUAT MONOMER peak was assigned in sample chromatograms by comparison to that of working standards. In blank sample chromatograms no peak appeared at the retention time of TRIQUAT MONOMER and, therefore, it was confirmed that only bi-distilled water was administered to the control animals.

The application formulations investigated during the study were found to comprise TRIQUAT MONOMER in the range of 90.8% to 99.4%, meeting the required content limit of ±20% with reference to the nominal concentration. Because single results found did not deviate more than 2.2% (<15%) from the corresponding mean, TRIQUAT MONOMER was considered to be homogeneously distributed in the preparations. In addition, the test item was found to be stable in application formulations when kept 7 days at room temperature due to recoveries which met the variation limit of 10% from the time-zero
(homogeneity) mean.
In conclusion, the results indicate the accurate use of the test item TRIQUAT MONOMER and bi-distilled water as vehicle during this study. Application formulations were found to be homogeneously prepared and of sufficient formulation stability under storage conditions used.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
other: dose adjusted for content of the main constituent
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
>= 2 420 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
Dose for the substance as registered (including residual water necessary for the stability)
Sex:
male/female
Basis for effect level:
other: See comments above

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, 100 mg/kg body weight/day of TRIQUAT MONOMER was established as the no-observed-effect-level (NOEL) in females, 1000 mg/kg body weight/day of TRIQUAT MONOMER was established as the no-observed-effect-level (NOEL) in males, and 1000 mg/kg body weight/day of TRIQUAT MONOMER as the no-observed-adverse-effect-level (NOAEL) for females (dose levels corrected for purity).
Executive summary:

Oral administration of TRIQUAT MONOMER to Wistar rats at doses of 100, 300 and 1000 mg/kg/day, for 28 days resulted in no deaths, no clinical signs of toxicological relevance, no effects upon the parameters of the functional observational battery (including no changes in the mean fore- and hindlimb grip strength), no effects upon food consumption and no toxicologically relevant differences in mean body weight, no effects upon the frequency or duration of estrus, and no adverse effects upon the parameters of hematology or clinical biochemistry. The mean absolute and relative organ weights were unaffected by the treatment with TRIQUAT MONOMER and there were neither macroscopical nor microscopical alterations of morphology that would indicate a relationship with the test item.

 

Test item-related findings were restricted to very slight and transient elevations in the mean locomotor activity in females treated with 300 mg/kg/day and 1000 mg/kg/day. However, these differences were reversible within the 60-minute observation period and were not evident in the males at these dose levels. Therefore, the differences were considered to be not adverse.

 

Based on the results of this study, 100 mg/kg body weight/day of TRIQUAT MONOMER was established as the no-observed-effect-level (NOEL) in females, 1000 mg/kg body weight/day of TRIQUAT MONOMER was established as the no-observed-effect-level (NOEL) in males, and 1000 mg/kg body weight/day of TRIQUAT MONOMER as the no-observed-adverse-effect-level (NOAEL) for females.