1,3-Propanediaminium, N1-[3-[[2-[dimethyl[3-[(2-methyl-1-oxo-2-propen-1-yl)amino]propyl]ammonio]acetyl]amino]propyl]-2-hydroxy-N1,N1,N3,N3,N3-pentamethyl-, chloride (1:3)

Endpoint summary

Description of key information

Based on a slight and transient increased locomotor activity observed in females treated with 300 or 1000 mg/kg/day (with no statistical significance), the NOAEL was considered to be 1000 mg/kg bw/day in females, and a NOEL was established at 1000 mg/kg bw/day in males (doses are corrected for the purity of the main constituent).
The NOAEL expressed for the substance as registered (i.e. with the residual water content necessary to the stability, and impurities) is therefore 2420 mg/kg bw/day. No target organ identified.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

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

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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.

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

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

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

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.

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

Critical effects observed:

not specified

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.

Reference 2

Endpoint:

repeated dose toxicity: oral

Remarks:

other: Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2009-12-17 to 2010-07-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study has been performed according to the OECD guideline No. 421, and in compliance with the Good Laboratory Practice. There were no circumstances that may have affected the quality or integrity of the data. All the parameters of a standard 28-day repated dose toxicity study were not examined in this study as this is a screening study for reproductive/developmental effects (see study C46073).

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

This is not a full repeated dose toxicity test but a screening for reproduction and developmental parameters. However, males and females were treated for a minimum of 28 days, and a number of general toxicity parameters were assessed (food consumption organ weights, histopathology).

GLP compliance:

yes (incl. QA statement)

Remarks:

In compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted May 18th, 2005 [SR 813.112.1]. This Ordinance is based on the OECD Principles of GLP, as revised in 1997 and adopted on Nov. 26th, 1997 [OECD Council C(97)186/Final].

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Animals: Rat, HanRcc: 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: 40 males (10 per group) and 40 females (10 per group)
Age (at Start of Treatment): 11 weeks
Body Weight Range (at Start of Treatment): Males (301 to 330 g) and females (174 to 206 g)
Identification: Cage card and individual animal number (ear tattoo).
Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
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: 18.8 – 71.8%). There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
Diet: Pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum (batch no. 60/09).
Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

DOSE FORMULATIONS
The dose formulations were prepared weekly using the test item as supplied by the Sponsor and using a correction factor of 2.42 so the dose actually ingested corresponds to the dose of the main constituent.

TRIQUAT MONOMER was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS
Dose formulations were stored in refrigerator (2 - 8 °C) in glass beakers.

Based upon the results of stability analyses performed within the Harlan Laboratories study C46062 (non-GLP Study), dose formulations were stable for at least one week.

TREATMENT
Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
Frequency of Administration: Once daily
Target Dose Levels (expressed as the dose of the main constituent): Group 1: 0 mg/kg/day (control group); 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 28-Day Oral (Gavage) toxicity study in Han Wistar, Harlan Laboratories Study C46073, using dose levels of 100, 300, and 1000 mg/kg/day, resulting in a NOAEL of 1000 mg/kg/day.
Dose Volume: 10 mL/kg body weight
Duration of Acclimatization Period: 7 days
Duration of Treatment Period: Males (minimum 4 weeks); females (approximately 7 weeks)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF DOSE FORMULATIONS
On the first treatment day samples from 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 concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (3 hrs and 7 days). Before the end of treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Analytic Department (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by HPLC coupled to an UV detector following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.

Samples were considered accurately prepared and sufficiently stable if the following acceptance criteria were met: ±20% of nominal for sample content, ±15% deviation from mean calculated from top, middle and bottom samples for homogeneity and ±10% from time-zero reference (content or mean of homogeneity samples).

Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

The application formulations investigated during the study were found to comprise TRIQUAT MONOMER in the range of 83.2% to 100.6% and, thus, the required content limit of ±20% with reference to the nominal concentration was met. The homogeneous distribution of TRIQUAT MONOMER in the preparations was approved because single results found did not deviate more than 7.2% (<15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept 7 days in the refrigerator (2 - 8 °C) due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

Duration of treatment / exposure:

Males: Minimum 4 weeks
Females: Approximately 7 weeks

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Acclimatization: 7 days (males and females)
First Test Item Administration : Day 1 of pre-pairing (males and females)
Pre-Pairing: 14 days (males and females)
Pairing: 15 days maximum (females); 15 days (males)
Gestation: approximately 21 days (females)
Treatment Ends: On day 3 post partum (females), on day before sacrifice (males)
Necropsy: On day 4 post partum (females); after a minimum of 28 days treatment (males)

Observations and examinations performed and frequency:

Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.
Food Consumption: Males (weekly during pre-pairing and after pairing periods; females (pre-pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum). No food consumption was recorded during the pairing period.
Body Weights: Recorded daily from treatment start to day of necropsy.

Sacrifice and pathology:

TERMINATION OF THE STUDY

Males were sacrificed after they had been treated for at least 28 days. Dams were sacrificed on day 4 post partum.

If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

NECROPSY

All parent animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

At the scheduled sacrifice, all parent animals were killed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

For the parent animals, special attention was directed at the organs of the reproductive system.
The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

ORGAN WEIGHTS

At the scheduled sacrifice, the testes and epididymides of all parental males were weighed as pairs.

TISSUE PRESERVATION

The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.

The testes and epididymides from all parental males were preserved in Bouin’s fixative. The prostate and seminal vesicles from all males were fixed in neutral phosphate buffered 4% formaldehyde solution.

In addition, all organs showing macroscopic lesions of all adult animals were preserved.

HISTOTECHNIQUE

All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the study pathologist.

HISTOPATHOLOGY

Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist.

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Histological examination of ovaries was carried out on one female (no. 51) that did not give birth. In addition, microscopic examination of the reproductive organs was made in one infertile male (No. 11).

A peer review was carried by a pathologist (Harlan Laboratories Ltd, Switzerland).

Other examinations:

LITTER OBSERVATIONS
The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.


TERMINATION AND NECROPSY OF OFFSPRING

Pups were sacrificed on day 4 post partum.

All pups sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

At the scheduled sacrifice, all pups were killed by an injection of sodium pentobarbital.

Dead pups, except those excessively cannibalized, were examined macroscopically.

All pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- 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 was applied if the variables could be dichotomized without loss of information.

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

Description (incidence and severity):

Slightly lower mean food consumption at 1000 mg/kg/day in females during the lactation period (not statistically significant): not considered adverse (no related effect on mean body weight).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In females at 300 mg/kg, dark-red nodules on left uterine horn and hemorrhagic vagina were noted in one female. This was not considered to be test item-related.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All findings recorded were within the range of normal background which may be recorded in animals of this strain and age.

Histopathological findings: neoplastic:

not examined

Details on results:

1 IN-LIVE DATA PARENTAL ANIMALS

1.1 MORTALITY
All animals survived until the scheduled necropsy.

1.2 CLINICAL SIGNS OR OBSERVATIONS
No clinical signs or symptoms were observed during the study.

1.3 FOOD CONSUMPTION OF MALES
Pre-pairing Period
No statistically significant alterations were observed in mean food consumption.

In order of ascending dose levels, the overall differences in food consumption were: 2.1%, -3.9% and -5.0% during the pre-pairing period (percentages refer to the respective values of the control group). These differences were not meaningful as the food consumption was stable in all groups over the pre-pairing period.

1.4 FOOD CONSUMPTION OF FEMALES
Pre-pairing, Gestation and Lactation Periods

No statistically significant alterations of mean food consumption were observed at any dose level when compared to the respective values in the control group during the pre-pairing, gestation and lactation periods.

In order of ascending dose levels, the overall differences in food consumption were: 3.2%,
-2.2% and +1.1% during the pre-pairing period, +2.6%, +0.9% and +3.1% during the gestation period and ±0.0%, -5.7% and -12.2% during the lactation period (percentages refer to the respective values of the control group). Although the mean food consumption was lower during the lactation period, body weights were not affected. Thus no adverse effect occurred.

1.5 BODY WEIGHTS OF MALES
Pre-pairing and Pairing Periods:
No statistically significant alterations were noted in mean body weight and mean body weight gain of males throughout the entire duration of the study.

In the order of ascending dose levels, the overall mean body weight gains were: +15%, +14%, +13% and +14% during the pre-pairing period, +9%, +10%, +8% and +8% during the pairing period (percentages refer to the respective time intervals).

1.6 BODY WEIGHTS OF FEMALES
Pre-pairing, Gestation and Lactation Periods:
No statistically significant alterations of mean body weights and mean body weight gain were observed at any dose level when compared to the respective values in the control group.

In the order of ascending dose levels, the overall mean body weight gain was +9%, +7%, +8% and +7% during the pre-pairing period, +60%, +61%, +59% and +56% during the gestation period and +4%, +4%, +3% (0 to 21 days) and +4% during the laction period (percentages refer to the respective time intervals).


2 REPRODUCTION AND BREEDING DATA
See details in Summary of the OECD421 in Section 7.8.1


3 TERMINAL FINDINGS - PARENTAL ANIMALS

3.1 ORGAN WEIGHTS
In males, weights (absolute and relative to body weight) of testes and epididymides in all test item-treated groups were comparable to those of the control groups and thus were not affected by the treatment with the test item.

3.2 MACROSCOPICAL FINDINGS
No abnormal findings were noted in males. In females, the only findings observed were dark red firm nodules on left uterine horn and hemorrhagic vagina in one female (No. 68) in group 3 and cervix with gray white mucus and right horn dilated in one female in group 1 (No. 44). Type and incidence of these findings did not indicate any test item-related effect.

3.3 HISTOPATHOLOGY FINDINGS
All findings recorded were within the range of normal background, which may be recorded in animals of this strain and age. No test item-related histological findings were recorded in ovary of one female (no. 51), which did not give birth and in reproductive organ of one infertile male (no. 11).

No differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals.

Dose descriptor:

NOEL

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: No reproduction or developmental toxicity.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

other: Dose adjusted for the main constituent content.

Sex:

male/female

Basis for effect level:

other: In the absence of any signs of general toxicity (the slightly lower mean food consumption observed in females during lactation period was not considered adverse since the mean body weight was not affected).

Dose descriptor:

NOAEL

Effect level:

2 420 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

Dose of substance as registered (including residual water necessary for the stability)

Sex:

male/female

Basis for effect level:

other: In the absence of any signs of general toxicity (the slightly lower mean food consumption observed in females during lactation period was not considered adverse since the mean body weight was not affected).

Critical effects observed:

not specified

Conclusions:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item TRIQUAT MONOMER to rats. TRIQUAT MONOMER was administered in Milli-Q-Water as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. TRIQUAT MONOMER was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

In absence of any adverse effect, the general NOAEL (No Observed Adverse Effect Level) was established at 1000 mg/kg/day.

Under the conditions of this study, the NOEL (No Observed Effect Level) for reproduction/ developmental toxicity was considered to be 1000 mg/kg/day.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of TRIQUAT MONOMER on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition.

Four groups of 10 males and 10 females were treated by gavage with TRIQUAT MONOMER once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to day 4 post partum.

 

The following dose levels were used:

                       Group 1:                0 mg/kg body weight/day (control group)

                       Group 2             100 mg/kg body weight/day

                       Group 3:            300 mg/kg body weight/day

                       Group 4           1000 mg/kg body weight/day

 

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water).

 

The following results were obtained:

 

PARENTAL ANIMALS General Tolerability

All animals survived until the scheduled necropsy and no clinical signs were noted during the whole study.

 

Food Consumption

Mean food consumption was not considered to be affected by the treatment with the test item in males and females. At 1000 mg/kg/day, the lower mean food consumption observed in females during lactation period was not considered to be adverse since mean body weight was not affected.

 

Body Weights

Mean body weight and body weight gain were not affected in males and females for the whole duration of the treatment.

 

Reproductive Data

Mating performance, fertility, duration of gestation, mean number of corpora lutea, of implantations and post-implantation losses were not adversely affected by the treatment with the test item.

 

Organ Weights

Mean weight of testes and epididymides of the test item treated groups were compared to that of control and thus not affected by the treatment with the test item.

 

Macroscopical Findings and Histopathological Examinations

No test item-related findings were observed during the macroscopical and histological examination.

 

Sperm Staging

No differences on the completeness of stages or cell populations of the testes were recorded between controls ad high dose animals.

 

Litter Data

The number of live pups at first litter check and the mean litter size was unaffected by treatment with the test item. Mean pup weight at first litter check and on day 4 post partum was not affected.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 420 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Study in compliance with current OECD guidelines and GLP standards.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No experimental data are currently available for the dermal and the inhalation routes.

The repeated treatment by the oral route produced a slight and transient increase in locomotor activity (not statistically significant) in females treated at 300 or 1000 mg/kg/day (dose corrected for the substance purity). No effects were observed in males.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
No significan adverse effects were observed at the dose 1000 mg/kg/day (dose as expressed for the main constituent), thus the NOAEL was at or above 1000 mg/kg bw/day (as expressed for the main constituent), i.e. 2420 mg/kg bw/day for the substance as registered (including the water preserving the stability)

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
see justification in endpoint record.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
see justification in endpoint record.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
No local effects observed in the acute dermal toxicity study nor in the skin irritation study.

Justification for classification or non-classification

No adverse effects were observed in the 28-d study by the oral route in rats, and no target organs were identified. The NOAEL was established at 2420 mg/kg bw/day for the substance as registered described in section 1.2. No classification is triggered.