Ethanaminium, 2-hydroxy-N-(2-hydroxyethyl)-N,N-dimethyl-, esters with C16-18 and C18-unsatd. fatty acids, chlorides

Endpoint summary

Description of key information

One 28 day repeated dose study comparable to OECD Guideline 407 and one sub-chronic toxicity study comparable to OECD Guideline 408 are available for the oral route of administration. The no effect level (NOEL) was determined to be 500 mg/kg/day.

In addition, no adverse effects were observed in an EOGRTS with basic test design up to the limit dose of 1000 mg/kg bw/d. This study included a thorough examination of general toxicity. Therefore, the NOAEL obtained in the EOGRTS is considered as the key value for hazard assessment.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-10-30 to 1993-02-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): N,N-dimethyl-2-(stearoyloxy)-N-[2-(stearoyloxy)ethyl]ethanaminium chloride in 10% aqueous dispersion.
- Physical state: liquid

Species:

rat

Strain:

other: Crl CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 4 weeks
- Weight at study initiation: 180-210 g (males) and 162-191 g (females)
- Fasting period before study: None
- Housing: individually in wire-mesh cages (for the first two days of the conditioning period, animals were housed 3/cage to allow for acclimation to the automatic watering system).
- Diet (e.g. ad libitum): ground Certified Rodent Chow #5002, Purina Mills, Inc., St. Louis, MO ad libitum
- Water (e.g. ad libitum): ad libitum, water was analyzed on a quarterly basis for the presence of heavy metals, pesticides and other contaminants
- Acclimation period: 15 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 73.2 +/- 1.0
- Humidity (%): 47.9 +/- 5.8
- Air changes (per hr): Not available
- Photoperiod (hrs dark / hrs light): 12 hours dark/light


IN-LIFE DATES: From: 1992-10-21 To: 1993-02-04

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was prepared in oral dosing suspensions at concentrations of 0.01, 0.1 and 5.0 % to provide dosage levels of 1, 10, and 500 mg/kg/bw/day.

Dosing suspensions of the test substance required modification of the vehicle in order to produce a consistent pH of 2.5. According to the Sponsor, the stock test preparations were pH 2.15. Since the stock suspensions were to be diluted with the pH Control (pH 2.5) to produce the 5 % dosing suspension, minor adjustment of the pH Control (to slightly increase pH with small amounts of 1 N sodium hydroxide) were necessary to insure a final pH of 2.5 for the dosing suspensions. Pre-test experiments were performed with the stock test article suspensions and the pH Control to determine the necessary adjustment.

Following adjustment of the pH Control for use as a diluent, this modified pH Control was mixed with the stock test substance suspensions to produce a 5 % dosing suspension of test substance (high dose) with a pH of approx. 2.5. The exact proportions of stock test substance suspension to modified pH Control varied with variation of test substance in the stock test substance suspension supplied by the Sponsor. The mid-and low-dose suspensions (0.1 and 0.01 %) were then produced via serial dilution of the 5 % dosing suspension with unmodified pH Control. Each phase of the blending and serial dilutions utilized double-rinsed polypropylene equipment. Each suspension was mixed with a magnetic stirring device for a minimum of 15 minutes between steps. The pH of the suspensions was monitored throughout the mixing period.

Final dilutions permitted once daily administration at a rate of 10 ml/kg. Fresh suspensions were prepared as needed (approx. bi-weekly) and stored at ambient temperature. Test substance suspensions were agitated with magnetic mixers for a minimum of 10 minutes each day immediately prior to dosing.


DIET PREPARATION
- Rate of preparation of diet (frequency): Not available
- Mixing appropriate amounts with (Type of food): Not available
- Storage temperature of food: Not available


VEHICLE: deionized water
- Justification for use and choice of vehicle (if other than water): Not available
- Concentration in vehicle: Not available
- Amount of vehicle (if gavage): Not available
- Lot/batch no. (if required): Not available
- Purity: Not available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance were collected on study days 25, 46, 77, and 92 for stability analysis; and on study days 25, 49, and 77 for concentration analysis. Duplicate samples were taken on days 25, 46, 63, 77, and 92 for microbiological characterization. All samples were delivered for analysis to the Sponsor. Stability and concentration samples were sent frozen and microbiological characterization samples were sent at ambient temperatures. All sampling was conducted immediately after active agitation of the suspensions.

pH TESTING: Dosing suspenstions and the pH Control solution were tested for conformance with pH requirements (pH 2.5 +/- 0.3) each day prior to dosing.

STABILITY ANALYSIS: These samples were collected prior to changeover to fresh test substance and dosing suspensions. They represent material and preparations in use for the preceding, approximate bi-weekly period. Samples were shipped frozen.

CONCENTRATION ANALYSIS: Samples of all dosing preparations and controls were taken on study days 1, 26, 49 and 77 for concentration analysis by the Sponsor. These samples were collected from freshly produced dosing suspensions prepared from recently received test material. Samples were shipped frozen.

MICROBIOLOGICAL CHARACTERIZATION: Samples of the dosing preparations and controls were taken on study days 25, 46, 63, 77, and 92 for microbiological characterization by the Sponsor. Similar to stability samples, those taken for microbiological characterization represented test substance and preparations in use for the proceeding, approximate bi-weekly period. These samples were shipped at ambient temperatures.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily, 7 days/week, for 13 weeks

Dose / conc.:

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

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

15 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Oral gavage was used to ensure delivery of a controlled and consistent dose of the test substance. Dosage levels were selected based on data generated from a 14-day (range-finding) toxicity study in rats (IRDC 191-1542), and the Sponsor's experience with subchronic
studies for structurally similar compounds. The volume administered to each rat (10 ml/kg) was adjusted based on the most recent body weight.
- Rationale for animal assignment (if not random): Prior to randomization into study groups, the animals were weighed, sexed and examined for evidence of disease and other physical abnormalities. After eliminating animals based on these criteria, animals found to be acceptable for study use
were randomized utilizing a block randomization procedure in which animals were stratified by body weight. Homogeneity of group variance by
body weight was used as the criterion for acceptance, at which time the randomization was accepted and permanent animal numbers were assigned.
- Rationale for selecting satellite groups: NA
- Post-exposure recovery period in satellite groups: NA
- Section schedule rationale (if not random): NA

Positive control:

No data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (mortality)
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Rats were observed twice daily for signs of overt toxicity at the times of the mortality/morbidity checks. Detailed clinical examinations were also performed at least weekly and included evaluations of appearance and condition, behaviour and activity, excretory function, respiration, orifices, eyes and palpable masses.


BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were obtained during the pretest period and weekly during the study and prior to termination.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
-Individual food consumption was calculated weekly during the study.


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:N/A


OPHTHALMOSCOPIC EXAMINATION: Yes (performed by a veterinary ophthalmologist using a Keeler Indirect Ophthalmoscope of the cornea, sclera, iris and fundus)
- Time schedule for examinations: during acclimation period and prior to terminal sacrifice
- Dose groups that were examined: all animals


HAEMATOLOGY: Yes
- Time schedule for collection of blood: taken at study termination
- Anaesthetic used for blood collection: Yes (CO2). Samples obtained via cardiac puncture
- Animals fasted: Yes, overnight. Animals had free access to water prior to blood collection. Urine was collected during the fasting period.
- How many animals: 10 animals/sex/group at termination. Studies from 6 animals scheduled for coagulation testing clotted prior to analysis. Blood samples were collected from 6 additional animals for coagulation testing in order to complete the 10/animals/sex/group protocol requirement.
- Parameters examined: leukocyte count, erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets, differential leukocyte count, prothrombin time (PT), activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: taken at study termination.
- Animals fasted: Yes, overnight. Animals had free access to water prior to blood collection. Urine was collected during the fasting period.
- How many animals: 10 animals/sex/group at termination. Studies from 6 animals scheduled for coagulation testing clotted prior to analysis. Blood samples were collected from 6 additional animals for coagulation testing in order to complete the 10/animals/sex/group protocol requirement.
- Parameters examined: sodium, potassium, chloride, calcium, inorganic phosphorous, alkaline phosphatase, total bilirubin, gamma glutamyl transpeptidase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen, creatinine, total protein, albumin, cholesterol, glucose.


URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected during the fasting period before blood was drawn.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: specific gravity and volume.


NEUROBEHAVIOURAL EXAMINATION: No



OTHER: Not available

Sacrifice and pathology:

All animals received a complete postmortem examination under the direct supervision of a pathologist. All animals were necropsied in a replicate order, and a necropsy body weight was obtained immediately following euthanization, but before exsanguination. This weight was used to determine the organ to body weight ratios. All animals were euthanized by methoxyfluorane anesthesis.

GROSS PATHOLOGY: Yes
After a thorough external examination, the skin was reflected from ventral midline incision and any abnormalities were identified and correlated with antemortem findings. The abdominal, thoracic and cranial cavities were examined for abnormalities and the organs removed, examined and, where required by protocol, placed in phosphate-buffered neutral formalin. All macroscopic abnormalities were recorded on the Pathology Record sheet.

Organ weights were determined for the following tissues for all animals and appropriate weight ratios calculated (absolute and relative to body and brain weights). Paired organs were weighed together. Adrenal, brain with stem, kidney, liver, ovary, testis.

HISTOPATHOLOGY: Yes
Representative samples of protocol designated organs and tissues were processed and embedded in paraffin for the preparation and microscopic examination of stained sections for all animals in the pH Control and 500 mg/kg/bw/day dosage groups.

A four-step grading system of trace, mild, moderate, and severe was used to define gradable lesions for comparison between dosage groups. Representative samples of the following tissues were collected and examined microscopically: adrenal, aorta, bone marrow smear, brain, exoribital lacrimal gland, eye including optic nerve, femur, GI tract, gonads, gross lesions, heart, kidney, liver, lung with bronchi, lymph nodes, mammary gland (females), pancreas, pitituary, prostate and seminal vesicle, salivary gland, sciatic nerve, skeletal muscle (thigh), skin, spinal cord, spleen, sternum (with bone marrow), thymus, thyroid, parathyroid, trachea, urinary bladder, uterus and cervix, vagina.

Other examinations:

Not available

Statistics:

Body weight, food consumption, clinical pathology laboratory, and organ weights (absolute and relative) were analyzed using analysis of variance (one-way classification) and Bartlett's test for homogenecity of variance. Treatment groups were compared to the pH Control group, by sex, using appropriate t-statistic (for equal or unequal variance) as described by Steal and Torrie. Dunnett's multiple comparison tables or pairwise comparisons with a Bonferroni correction were used to determine the significance of differences. Non parametric analyses were conducted as appropriate by transforming the data into ranks prior to analysis as described by Conover and Iman. All statistical analyses were performed with P less than or equal to 0.05 and P less than or equal to 0.01 used as levels of significance.

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related signs of overt toxicity were observed during the study. Clinical examinations of the animals did not produce any evidence of test substance-related effects.

Mortality:

no mortality observed

Description (incidence):

All rats survived to study termination.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant differences in body weights or body weight increases occurred over the course of the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No significant differences in food consumption occurred over the course of the study in any of the groups.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test substance-related ophthalmoscopic abnormalities were detected; the observations noted were representative of pathology that would be expected for this group of rats considering age, sex and strain.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Mean MCV values were statistically significantly lower (p less than or equal to 0.05) in male rats in the water control, 10 mg/kg/bw/day, and 500 mg/kg bw/day treatment groups as compared to the pH Control group. The mean MCV values in these groups were within +/- 2 standard deviations of this laboratory's historical control mean value for this parameter for rats of this strain, age, and sex. The mean MCV values in these groups also fall within published reference ranges for rats of this strain, age, and sex. Because of these considerations, because this change was not present in both sexes, and because this change also occurred in the water control group, this haematological change, although statistically significant, was not considered to be toxicologically significant or test article-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Mean urea nitrogen values were statistically significantly lower (p less than or equal to 0.01) in male rats in the 500 mg/kg bw/day treatment group as compared to the pH Control group. The mean urea nitrogen value in this group was within +/- 2 standard deviations of the testing laboratory's historical control mean value for this paramater for rats of this strain, age, and sex. Because of the small magnitude of the change, because this change was not present in both sexes, and because no other clinical, biochemical, or pathologic alterations correlating with decreased urea nitrogen were observed, this serum biochemical change, although statistically significant, was not considered to be toxicologically significant or test substance-related.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no changes that were considered to be toxicologically significant or test substance-related.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related changes in the mean absolute weights or mean relative organ weight ratios for the brain, adrenal, kidney, liver and testis of male and female rats.

There was a statistically significant decrease in the mean ovary/body weight ratio for female rats in the 500 mg/kg/bw/day group when compared with the pH Control. This ratio change was, at least in part, due to the variability in the mean body weights with the pH Control being the lowest of all the female groups and the 500 mg/kg bw/day group being the highest of the female groups on the study. There were no statistically significant changes in the mean absolute ovary weight and the ovary/body or the ovary/brain weight ratios when the 1, 10 and 500 mg/kg bw/day treatment groups were compared with the water control or when the same parameters of the ovary from the water control group were compared to the pH Control. The mean absolute ovary weight of the 500 mg/kg bw/day treatment group falls within the range of mean absolute ovarian weight fo 13-week CD rat studies at the testing laboratory. For these reasons, the decreased mean ovary/body weight ratio observed in this study was considered not to be biologically relevant and not test substance-related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related macroscopic findings in either male or female rats necropsied 13 weeks following oral intubation with Water or pH Control material or with 1, 10 or 500 mg/kg bw/day of test article.

A few macroscopic findings were noted at the terminal necropsy. The majority of these macroscopic findings were confirmed microscopically. These findings were considered to be incidental and usual for rats of this age and strain and not test article-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test substance-related microscopic findings in either male or female rats necropsied following 13 weeks oral intubation.

All macroscopic observations (except cystic ovary, # 41229) correlated with microscopic findings. There were several microscopic findings. These findings were considered to be incidental and usual for rats of this strain and age. The incidence of a given lesion was small and/or occurred with equivalent frequency in treated animals versus controls. There was no evidence of any infectious disease present in any of the rats that would adversely affect the results of the study.

The tissues available for microscopic examination were of satisfactory quality and quantity to adequately evaluate this study. Relevant in-life and necropsy data were available to the study pathologist and were considered in interpretation of the pathology findings. All reference to pathology interpretations in the final report were consistent.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Absolute and relative organ weights of ovary and testis and histopathology of reproductive organs (gonads, mammary gland (females only), prostate and seminal vesicle, uterus with cervix and vagina) revealed no test substance related findings.

Dose descriptor:

NOAEL

Effect level:

> 500 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: There were no significant adverse test substance-related effects in this study for any of the parameters measured.

Critical effects observed:

no

Statistically significant lower mean MCV values were noted between the 10 mg/kg bw/day, 500 mg/kg bw/day males and the water control male group when compared to the pH control water male group. These differences were not considered biological or test substance related as there were within + or - two standard deviations of the laboratory historical control mean values for rats of this strain, age and sex. In addition, they were within the standard published reference ranges.

The blood urea nitrogen values of the 500 mg/kg bw/day group was significantly lower than the pH control group in male rats. This differences were not considered test substance related as they were within + or - two standard deviatiosn of the laboratory historcal control mean values for rats of this strain, age and sex.

The ovary/body weight ratios of the 500 mg/kg bw/day female rats were signficant lower than the pH control females. The differences in the ovary/body weight ratios were not considered test substance related because of the variability in the mean body weights of pH control group, lowest of all female groups. In addition, there were no test related difference observed in the mean absolute ovary weight of the 500 mg/kg bw/day group in comparison to the other test or control groups and the mean ovary weight also fall with

within + or - two standard deviations of the laboratory historical control mean values for rats of this strain, age and sex.

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

Conclusions:

The test substance was administered orally via gavage daily to rats at dosage levels of 1, 10, and 500 mg/kg bw/day for a period of 13 weeks. No biologically significant adverse effects were observed in any test group. The NOAEL was determined to be the high dose level of 500 mg/kg bw/day.

Executive summary:

In a subchronic toxicity study comparable to OECD guideline 408, MDEA-Esterquat C16-18 and C18 unsatd.  (10 % a.i.) was administered to 15 Charles River CD rats / sex/ dose by gavage at dose levels of 1, 10 and 500 mg/kg bw/ day for a period of 13- weeks. One control group received the vehicle, deionized water, and a second control group received pH-adjusted, deionized water (pH 2.5). The regimen for both control groups was identical to treatment groups.

The following parameters were monitored during the study: clinical observations (detailed, weekly; mortality, morbidity, and overt signs of toxicity, twice a day); body weights (weekly); food consumption (weekly); clinical pathology (haematology, blood chemistry, and urinalysis; at termination); opothalmoscopic examinations (once pre-test and prior to sacrifice); macroscopic pathologic examination; absolute and relative organ weights; microscopic pathology.

There were no changes in any of these parameters that were considered to be toxicologically significant or test-substance related.

Reproductive parameters:

Absolute and relative organ weights of ovary and testis and histopathology of reproductive organs (gonads, mammary gland (females only), prostate and seminal vesicle, uterus with cervix and vagina) revealed no test substance related findings.

There is no evidence for specific target organ toxicity in this study.

The no effect level (NOEL) for this study is the high dose level of 500 mg/kg bw/day of the test article.

This subchronic toxicity study in rats is acceptable and satisfies to a large extent the guideline requirement for a subchronic oral study, with exception of highest tested dose. 500 mg/kg bw/day was tested as highest dose instead of 1000 mg/kg/bw/day as recommended by the actual OECD guideline 408.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-07-29 to 1992-08-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

27 July 1995

Deviations:

yes

Remarks:

highest recommended dose of 1000 mg/kg bw was not included in this study, not all necessary organ weights were taken e.g. heart, thymus, spleen were not evaluated

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): N,N-dimethyl-2-(stearoyloxy)-N-[2-(stearoyloxy)ethyl]ethanaminium chloride in 10% aqueous dispersion
- Physical state: liquid

Species:

rat

Strain:

other: Charles River CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI
- Age at study initiation: approximately 6 weeks of age at initiation of dosing
- Weight at study initiation: 161-195 g (males) and 131-156 g (females)
- Fasting period before study: Not available
- Housing: For the first 5 days of the conditioning period, animals were housed 3/cage to allow for acclimation to the automatic watering system. After this time, animals were housed individually in wire-mesh cages. Cage banks were rotated every two weeks.
- Diet (e.g. ad libitum): Certified Rodent Chow #5002, Purina Mills, Inc., St. Louis, MO ad libitum (except prior to clinical laboratory tests and necropsy, when food was removed)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days (animals were observed for any clinical signs of disease and all animals were given detailed physical and opthalmological examinations).


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 +/- 0.5
- Humidity (%): 56 +/- 5.7
- Air changes (per hr): Not available
- Photoperiod (hrs dark / hrs light): 12 hrs light/dark. (Light intensity measurements were conducted weekly).


IN-LIFE DATES: From: 1992-07-15 To: 1992-08-27

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
-Deionized water control group: the appropriate amount of DI water was dispensed into the appropriate number of polypropylene containers for each day of dosing and stored at room temperature. The pH was measured and recorded.
-Vehicle pH 2.5 DI water group: the appropriate amount of DI water was measured in a suitable container, stirred using a motor driven propeller and the pH was adjusted to 2.5 using 1 N HCl. After this, the appropriate amount of pH 2.5 DI water was dispensed into the appropriate number of polypropylene containers for each day of dosing and stored at room temperatures. The pH was measured and recorded.
-Test article group: The test article was mixed for at least 10 minutes prior to use using a motor driven propeller. For the 500 mg/kg/bw/day dosage level, the required volume of test article was measured, using polypropylene equipment and placed into a polypropylene beaker. The required amount of vehicle was measured to yield an appropriate amount of prepared test material. The mixture was stirred for at least 15 minutes using a magnetic stir bar and stir plate. The pH was measured and recorded. A portion of the 500 mg/kg/bw/day mixture was used to prepare the 10 mg/kg/bw/day dosage level mixture and a portion of the 10 mg/kg/bw/day mixture was used to prepare the 1 mg/kg/bw/day dosage level mixture in similar manner except a syringe was used for measurement of prepared test material. In each case, the test material was dispensed into the appropriate number of polypropylene containers for each day of dosing using a polypropylene syringe and was stored at room temperature. For one sample (Batch 1), fresh dosing solutions of each concentration were made every two weeks from the stock dispersion. For the other sample (Batch 2), fresh dosing solutions of each concentration were made on day 23.



DIET PREPARATION
- Rate of preparation of diet (frequency): N/A
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A


VEHICLE
- Justification for use and choice of vehicle (if other than water): Not available
- Concentration in vehicle: Not available
- Amount of vehicle (if gavage): Not available
- Lot/batch no. (if required): 7229201, 7279201, 7299201, 8109201, 8119201, 8179201, 8209204, 8249201, 8259201
- Purity: Not available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

-Stability and concentration analysis of dosing solutions was conducted by the Sponsor. Test substance "batch 1" (see analytical section above) was used on study up to day 22. From day 23 and thereafter, test substance "batch 2" was used.
-Samples of the test substance were collected on days 1, 8, 14, 21, 23, and 29 and were sent to the Sponsor for analysis. Samples of the dosing preparations were collected on study days 1, 14, 23, and 28 and sent to the Sponsor for microbiological characterization. Analytical samples were shipped in polypropylene vials with polyethylene lids (on dry ice).

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Daily, 7 days/week for 4 weeks

Dose / conc.:

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

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Oral gavage was used to ensure delivery of a controlled and consistent dose of the test material.
- Rationale for animal assignment (if not random): Prior to randomization into study groups, the animals were weighed, sexed, and examined for evidence of disease and other physical abnormalities. After eliminating animals based on these criteria, animals found to be acceptable for study use were randomized utilizing the Xybion procedure as follows: For each sex, the temporary animal numbers and corresponding body weights were entered onto a magnetic disk. At the end of the pretest period, animals were assigned to dose groups and permanent animal numbers were assigned using the following computer-calculated randomization procedure: A computerized sort developed a listing in order of descending body weight which was reduced to the number of animals required for the study by alternately discarding first the lightest, then the heaviest animal. This reduced list was blocked into a blocks of x animals, where the number of blocks was equivalent to the number of groups. Animals in each block were then distributed among groups by means of a computer-calculated lists of x random numbers. Homogeneity of group variance by body weight was used as the criterion for acceptance. If the group variance were judged heterogeneous, new randomizations were applied until homogeneity was established, at which time the randomization was accepted and permanent animal numbers were assigned. All unassigned rats were euthanized.
- Rationale for selecting satellite groups: NA
- Post-exposure recovery period in satellite groups: NA
- Section schedule rationale (if not random): NA
- Species selection rationale: The rat is a standard laboratory animal used to evaluate the subchronic toxicity of new chemicals. Considerable control data have been accumulated.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Rats were observed for moribundity and mortality at least twice daily throughout the study.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Rats were observed twice daily for signs of overt toxicity at the time of the moribundity/mortality checks. Detailed observations of appearances and condition, behaviour and activity, excretory functions, respiration, orifices, eyes and palpable masses were conducted at least once weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were obtained pretest, and weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Individual food consumption measurements were determined weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during the pretest period and toward the end of the study (day 23).
- Dose groups that were examined: all rats.
- Conducted by a vetarinary opthalmologist. Opthalmoscopic examation of the eye was performed following pupillary dialation with 1% tropicamide solution. A binocular indirect ophthalmoscope was utilized with a positive 20 diopter focusing and magnifying lens. This instrumentation allows for the evaluation of the ocular tissues with steroscopic vision at a magnification of approx. 4-5 power. The clarity of the ocular media (precorneal tear film, cornea, aqueous humor, lens and vitreous humor) and fundic reflex were initially evaluated. The ocular adnexa and iris were viewed under magnification and the lens focal distance changed to view the fundic tissues (approx. the equator of the globe). A direct or slit lamp examination was performed when the ophthalmologist determined it was necessary to clarify a particular finding.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Not available
- Anaesthetic used for blood collection: No. Blood samples were obtained from the abdominal aorta.
- Animals fasted: Yes (overnight). Animals had free access to water.
- How many animals: 10 animals per sex/ group
- Parameters examined: Leukocyte count, erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets, differential leukocyte count, prothrombin time (PT), activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Not available
- Animals fasted: Yes (overnight). Animals had free access to water.
- How many animals: 10 animals per group.
- Parameters examined: sodium potassium, chloride, calcium, inorganic phosphorous, alkaline phosphatase, total bilirubin, gamma glutamyl transpeptidase (GGT), aspartate aminotransferase (AST), alanine amino transferase (ALT), urea nitrogen, creatinine, total protein, albumin, globulin, cholesterol, and glucose.


URINALYSIS: Yes
- Time schedule for collection of urine: collected during the fasting period before blood collection.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: volume and specific gravity.


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Ten animals/sex/group received a complete postmortem examination under the direct supervision of a pathologist. The animals were euthanized by methoxyfluorourane anesthesia followed by exsanguination and necropsied in random order. The 15 animals/sex/group which remained after the interim necropsy were sacrificed without necropsy and discarded due to the early termination of the study.

After a thorough external examination, the skin of each animal was reflected from a ventral midline incision and any subcutaneous abnormalities identified and correlated with antemortem findings. The abdominal, thoracic and cranial cavities were examined for abnormalities and the organs removed, examined, and where required by protocol, placed in phosphate-buffered neutral formalin (except the eyes) at a dilution of 1 part tissue to at least 10 parts fixative. The eyes were collected in Davidson's fixative. The pituitary was fixed in toto. All macroscopic abnormalities were recorded on the Pathology Record sheet.

A body weight was obtained for each animal following euthanasia and prior to exsanguination for use in establishing relative organ weights. Protocol designated organs were trimmed free of fat and connective tissue and weighed. Organ weights from all animals were determined and appropriate weight ratios calculated (absolute and relative to body and brain weights). Paired organs were weighed together. The following organs were weighed: adrenal, brain (with stem), kidney, liver, ovary, testis.


HISTOPATHOLOGY: Yes
Representative samples of protocol designated organs and tissues were processed in paraffin for the preparation and microscopic examination of haematoxylin- and acsin-stained sections. A full complement of organs and tissues was prepared for all animals in the control and high dose groups. Only gross lesions were prepared for animals in the low and mid dosage groups. A four-step grading system of trace, mild, moderate and severe was used to define gradable lesions for comparison between dosage groups. The following list constitutes the protocol designated organs and tissues collected and examined microscopically: adrenal, aorta, bone marrow smear, kidney, liver, brain, exorbital lachrymal glands, eye including optic nerve, femur (including articular surface), GI tract, gonads, gross lesions, heart, lung with bronchi, lymph nodes, mammary gland (female only), pancreas, pituitary, prostate and seminal vesicle, salivary gland, sciatic nerve, skeletal muscle (thigh), skin, spinal cord, spleen, sternum (with bond marrow), thymus, thyroid/parathyroid, trachea, urinary bladder, uterus, vagina.

Other examinations:

Not available

Statistics:

Body weights, food consumption values, haematological, biochemical and urological parameters, and absolute and relative (to body and brain weights), organ weights were analyzed using analysis of variance (one-way classification) and Bartlett's test for homogeneity of variance.

Treatment groups were compared to the control group by sex, using appropriate t-statistics (for equal or unequal variance) as described by Steel and Torrie. Dunnett's multiple comparison tables or pairwise comparisons with a Bonferroni correction were used to determine the significance of differences. Nonparametric analyses were conducted as appropriate by transforming the data into ranks prior to analysis, as described by Conover and Iman. All statistical analyses were performed with p less than or equal to 0.05 and p less than or equal to 0.01 used as levels of significance.

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related clinical findings were observed in any animal during the study period. Signs observed were considered incidental or agonal in nature.

Mortality:

no mortality observed

Description (incidence):

All animals survived to study termination.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistical significance was noted.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Statistical significance was noted (primarily at week one of study) when comparing the 1 and 500 mg/kg bw/day dosage level animals to animals from both of the control groups. It was concluded that this was not test substance-related in the overall conclusions.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test substance-related opthalmoscopic abnormalities were detected; the observations noted were representative of pathology that would be expected for this group of rats considering age, sex and strain, except for three animals that exhibited choroiratinal scarring (focal scarring or retinal loss).

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males from the 500 mg/kg bw/day dosage group showed a slight but statistically significant decrease in the MCH index. However, since no corresponding change was detected in any other hematologic parameter, this change in the MCH was not considered test substance-related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No meaningful changes were detected in any parameter.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No meaningful changes were detected in any parameter.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test substance-related or statistically significant changes in the absolute or relative organ weights of male and female rats necropsied following 28 days of oral intubation with 1, 10, or 500 mg/kg bw/day of the test substance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related macroscopic observations in either male or female rats examined following 28 day oral intubation with 1, 10, or 500 mg/kg bw/day of the test substance. No deaths occurred during the course of the study.

The tan, macroscopic focus observed in the 1 mg/kg bw/day female rat was confirmed microscopically as a focal area of trace bronchopneumonia. The macroscopically enlarged mandibular lymph nodes, a common finding in the rat, were correlated microscopically with medullary plasmacytosis, reactive lymphoid follicules or lymphoid hyperplasia. The macroscopic skin abrasions, erosion and scab formations were located on the ventral aspect of the neck and correlated microscopically with ulcers in the skin. The ulcers were thought to be associated with rubbing of the skin of the neck on the edge of the opening in the feeder box.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no microscopic test substance-related findings in either male or female rats necropsied after 28 days oral intubation with 1, 10, and 500 mg/kg bw/day of the test article.

All macroscopic observations correlated with microscopic findings. The microscopic findings were considered to be incidental and usual for rats of this strain and age. The incidence of a given lesion occurred with equivalent frequency in treated versus controls. There was no evidence of any infectious disease present in any of the rats that would adversely affect the results of the study.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Reproductive parameters:
Organ weights of ovary and testis and histopathology of reproductive organs (gonads, mammary gland (females only), prostate and seminal vesicle, uterus and vagina) revealed no test substance related findings.

Dose descriptor:

NOAEL

Effect level:

> 500 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: No significant treatment-related effects were seen at the highest dose administered.

Critical effects observed:

no

Mean food consumption of the 1 and 500 mg/kg bw/day female groups were found to be significantly higher than the water control female group at week 1. No effects were observed in the subsequent weekly measures through week 4. These differences were not dose related and therefore were considered incidental and unrelated to the test substance.

The MCH of the 500 mg/kg bw/day males were found to be significantly lower than the water control males at week 4. These differences were considered incidental and unrelated to the test substances.

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

Conclusions:

The test substance, administered to rats via oral intubation daily for four weeks did not produce any test related toxicological responses up to levels of 500 mg/kg bw/day. The NOAEL was determined to be 500 mg/kg bw/day.

Executive summary:

In this Repeated Dose, 28-day Oral Toxicity Study performed comparable to OECD guideline 407, 12 May 1981 MDEA-Esterquat C16-18 and C18 unsatd.  (10 % a.i aqueous dispersion) was administered to groups of 25 Charles River CD rats male and female by oral gavage at dose levels of 1, 10 and 500 mg/kg bw/day for 28 days. Two other control groups received either deionised water or to pH 2.5 adjusted (HCL) deionised water (vehicle).

All animals survived to study termination. No test substance-related findings were detected or observed in clinical examinations, body weights, food consumption values, opthalmoscopic examinations, haematology, clinical biochemistry or clinical pathology evaluations. During the anatomical pathology examinations, no test substance-related findings were observed or detected during macroscopic examinations, organ weight evaluations or microscopic examinations.

Reproductive parameters:

Organ weights of ovary and testis and histopathology of reproductive organs (gonads, mammary gland (females only), prostate and seminal vesicle, uterus and vagina) revealed no test substance related findings.

There is no evidence for specific target organ toxicity in this study.

The NOAEL is ≥ 500 mg/kg bw/day in this study.

 

This Repeated Dose, 28-day Oral Toxicity Study in rats is acceptable and satisfies to a large extent the guideline requirements for a short-term repeated dose toxicity study OECD 407, with exception of highest tested dose. 500 mg/kg bw/ day was tested as highest dose instead of 1000 mg/kg bw/day as recommended by the actual OECD guideline 407.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

study currently in draft

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: P: 7-8 wks
- Weight at study initiation: (P) Males: 266 - 341 g; Females: 173 -243 g
- Housing:
From arrival to pairing: up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm.
During mating (P generation): one male to one female in clear polysulfone cages measuring approximately 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor.
After mating: males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm).
- Diet (e.g. ad libitum): laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019, SettimoMilanese (MI), Italy); ad libitum (except during the fasting procedure necessary for clinical pathology investigations)
- Water (e.g. ad libitum): ad libitum (except in the case of urinalysis investigations)
- Acclimation period: 18 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2°C
- Humidity (%): 55±15%
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was suspended in the vehicle. The preparations were made daily. Concentrations were calculated and expressed in terms of test item as supplied. The preparation of the test item included a very slow addition of the vehicle to the test item and a manual mixing followed by a magnetic mixing for at least 1 hour. The very slow addition of the vehicle and a low stirring speed of the preparations were necessary conditions in order to minimize the formation of air bubbles.

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in the range from 10 to 100mg/mL (r > 0.98; accuracy 85-115%; precision CV < 10%).
6 hour stability at room temperature was verified in the range from 10 to 100mg/mL.

Duration of treatment / exposure:

Males: 96-100 days (10 weeks prior to pairing, during mating period and thereafter until the day before necropsy)
Females: at least 85 days (at least 10 weeks prior to pairing, during mating, gestation and post partum periods until Day 21 post partum, the day before sacrifice)
The not pregnant females, humanely killed females, females with total litter loss and females that did not give birth were dosed up to the day before necropsy.

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

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

No. of animals per sex per dose:

25

Details on study design:

- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on information from a previous GLP compliant study (OECD TG 421).

Positive control:

n.a.

Observations and examinations performed and frequency:

Animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment (P generation only) and at least once a week from the start of treatment (Day 21 or 22 of age for Cohorts 1A and 1B) until termination
- Signs recorded included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations:
males: day of allocation (Day 21 of age for Cohorts 1A and 1B) and then at approximately weekly intervals from the first day of treatment
females: day of allocation (Day 21 of age for Cohorts 1A and 1B) and then approximatelyweekly from the first day of treatment until termination or until positive identification of mating (P generation)
Females of P generation after mating were also weighed on Days 0, 7, 14 and 20 post coitum and on Days 1, 4, 7, 14 and 21 post partum.
Body weight was also recorded in Cohorts 1A and 1B on the day when they attained puberty (completion of preputial separation or vaginal patency).
All animals were weighed on the day of sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
at weekly intervals starting from Day 1 of dosing and up to pairing for P generation and starting from nominal Day 28 for F1 generation
P males: at weekly intervals from the end of the mating period to termination
P females: on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 7, 14 and 21 post partum starting from Day 1 post partum.

WATER CONSUMPTION: No

OTHER:
Urinalysis (10 (random) animals/sex/group): P generation and Cohort 1A
- during last week of treatment, individual overnight urine samples were collected from 10 males and 10 females (for P generation females with viable litters), all randomly selected from each group (the same animals selected for clinical pathology investigation under the same condition) Before starting urine collection, water bottles were removed from each cage and each animal received approximately 10 mL/kg of drinking water by gavage.
- parameters: Appearance, Volume (manually recorded), Specific gravity, pH, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Blood, Epithelial cells, Leucocytes, Erythrocytes, Crystals, Spermatozoa and precursors, Other abnormal components

Blood clotting time (10 randomly selected animals/sex/group): P generation
During the last week of treatment, a measure of blood clotting time was performed once from 10 parental males and 10 parental females (females with viable litters), all randomly selected from each group.

Clinical pathology investigations: P generation and Cohort 1A
Males: Once during the last week of treatment, samples of blood for haematology and clinical chemistry were collected from the retro-orbital sinus under isoflurane anaesthesia, from 10 males randomly selected from each group, under condition of food deprivation.
Females: As a part of the sacrificial procedure, and under condition of food deprivation, blood samples for haematology and clinical chemistry were withdrawn under isoflurane anaesthesia from the abdominal vena cava, of 10 females, all randomly selected from each group.
Parameters:
Haematology: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelets
Coagulation: Prothrombin time, Activated partial thromboplastin time
Clinical chemistry: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma glutamyltransferase, Bile acids, Urea, Creatinine, Glucose, Triglycerides, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride, Phosphorus

Thyroid hormone determination (T3, T4 and TSH) (10 animals/sex/group): P generation and F1 pups
Males: Once during the last week of treatment, samples of blood for thyroid hormone determination were collected from the retro-orbital sinus under isoflurane anaesthesia, from 10 males randomly selected from each group, under condition of food deprivation. The timing of the blood collection for thyroid hormone determination was as close as possible between animals (within a time window of 1-4 hours) and at the same time of the day (within the same time window of 1-4 hours) in case of sampling on different days.
Females: As a part of the sacrificial procedure, and under condition of food deprivation, blood samples were withdrawn under isoflurane anaesthesia from the abdominal vena cava (for P generation females, with viable litters, if possible) of all females. The timing of the blood collection for thyroid hormone determination was as close as possible between animals (within a time window of 1-4 hours) and at the same time of the day (within the same time window of 1-4 hours) in case of sampling on different Days.
F1 pups (Days 4 and 21 or 22 post partum): On Day 4 post partum, as part of the necropsy procedure, blood samples (approximately 0.2 mL) were taken from 2 of the non selected pups (1 male and 1 female, if possible) of each litter per group if the number in the litters was sufficient. Blood samples were withdrawn under light ether anaesthesia from the heart by intracardiac puncture. Samples from different pups per sex was pooled if necessary. On Day 21 or 22 post partum, as part of the necropsy procedure, blood samples (approximately 0.5 mL) were taken from 2 pups (1 male and 1 female, if possible) if the number in the litters was sufficient.

Toxicokinetic studies:
Blood collection for detection of test item: P generation and F1 pups
Males: Blood samples (approximately 0.3 mL) were taken from the tail vein of 5 males/group during the last day of treatment at 2 time points (1 and 4 hours post-dose).
Females: Blood samples (approximately 0.3 mL) were taken from the tail vein of 5 females/group on Day 18 post coitum and Day 21 post partum at 2 time points (1 and 4 hours post-dose).

Milk collection for detection of test item: Dams
Milk samples were collected (at approximately 1 hour after dosing) from 5 females of each group, on Day 21 post partum (the same females selected for blood collection). On the day of collection oxytocin was administered to the selected females. Milk samples were collected under isoflurane anaesthesia. The samples were transferred into tubes and were frozen at -80°C, pending further information from the Sponsor.

Sacrifice and pathology:

SACRIFICE
- Male animals: All surviving animals (Males of the P generation were killed after the weaning of the majority of F1 females.)
- Maternal animals: All surviving animals (P generation: Females with live pups were killed on Day 22 post partum. The females with total litter loss were killed on the day of the occurrence of total litter loss or shortly after. The females showing no evidence of copulation were killed after 25 days of the last day of the mating session. The females which did not gave birth 25 days after positive identification of mating were killed shortly after.)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Dams and litters - of P generation
All dams with litters were sacrificed on Day 22 post partum. The uteri and ovaries of each female which gave birth were inspected for the following:
1. Number of corpora lutea
2. Number of implantation sites

HISTOPATHOLOGY / ORGAN WEIGHTS
The examination was performed as detailed below:
i Tissues specified in table 6.9.13 and 6.9.14 from all animals in the control and high dose groups
ii Tissues specified in table 6.9.13 and 6.9.14 from all animals in the low and medium dose groups killed or dying during the treatment period
iii Reproductive organs of animals suspected of reduced fertility (e.g. that failed to mate or conceive)
iv All abnormalities in all groups

Bone marrow collection - P generation and Cohort 1A
During the necropsy procedure, shortly after the death of each animal (except for those found dead), bone marrow samples were obtained from the femur of all animals (Parental generation and Cohort 1A).
The evaluation of bone marrow was performed in 10 male and 10 female animals per group, all randomly selected. A differential count was performed including calculation of the myeloid/erythroid cell ratio.

Organ weights - P and Cohorts 1A and 1B animals
- all animals completing the scheduled test period
- see table 6.9.13

Enumeration of ovarian follicles - P generation and Cohort 1A and 1B females: all animals in the control and high dose groups

Other examinations:

examinations related to reproductive parameters are described in iuclid section 7.8.1. Toxicity to reproduction

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n>5.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
Intergroup differences between the control and treated groups was assessed by the nonparametric version of theWilliams test. Details of all tests used and the data to which they were applied are included in the study report. The criterion for statistical significance was p<0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males
At daily clinical examination, no adverse clinical signs were observed in treated males.
In the control group, one male (no. X0180032) showed swollen ear and was isolated in cage for approximately 2 weeks.
In the low dose group, hunched posture, dyspnoea and piloerection were noted in one male (no. X0180064) found dead after 97 days of treatment. Scabs and scabs with hairloss were recorded in two males from the same group (nos. X0180082 and X0180084).
In the mid-dose group, hairloss (no. X0180120), tooth cut or broken (nos. X0180130 and X0180132), scabs (no. X0180136) and swollen ear (no. X0180140) were observed.
In the high dose group, salivation was recorded in all males starting from approximately 9 weeks of treatment. Other minor clinical signs, such as rales, damaged tail, scabs and hairloss were occasionally noted in few animals.

Females
At daily clinical examination, no adverse clinical signs were observed in treated females.
Kyphosis and/or pallor were recorded in one control female (no. X0180033) and one high dose female (no. X1080181) sacrificed on Days 19 and 26 post coitum, respectively.
The other observed minor clinical signs, such as hairloss, scabs, staining and damaged ear were of low incidence and sporadically distributed among the control, low and mid-dose groups.
In the high dose group, salivation was observed in most females after approximately 9 weeks of treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Males
One male (animal no. X1080064) of the low dose group was found dead on Day 27 of the mating phase (Day 97 of treatment). At post mortem examination, this animal showed a single firm mass in the thymus, enlarged and swollen spleen, dark and/or red colour of epididymides, jejunum and left testis. The histopathological evaluation revealed a malignant leukemia with metastases in the heart, kidneys, liver, lungs, prostate, spleen and thymus. The factor contributory to the death of this animal could be attributed to malignant leukemia.

Females
Two females were sacrificed for humane reasons: one female (no. X1080033) from control group and one female (no. X1080181) from high dose group.
Female no. X1080033 (control) was sacrificed on Day 19 post coitum (pregnant). At macroscopic observations, a single dark firmsubcutaneous mass associated with a single scab of the skin was observed. At histopathology, the most relevant changes were an adenocarcinoma in the mammary glands (which correlated to the subcutaneous mass observed during necropsy) and scab formation of the skin. These findings could be considered the factors contributory to the poor conditions of this animal.
Female no. X1080181 (high dose) was sacrificed on Day 26 post coitum with clinical signs of piloerection and pallor. At post mortem examination, brown staining of the muzzle and presence of dead foetuses in uterus were observed. The luminal dilatation of the uterus observed at histopathology was probably due to this condition. The presence of dead foetuses in uterus was considered the factor contributing to the illness status of this animal, suggesting that the animals had problem with the parturition.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Means of body weight of treated males and female were, in general, comparable to the control group throughout the study. The occasionally statistically significant increases noted in high dose females at the end of the mating phase and in mid-dose females during the post partum period were not adverse.
Means of body weight gain of treated males and females were, in general, comparable to the control group. The statistically significant increase or decrease noted occasionally was considered not adverse.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption in treated males and females was comparable to the control group during the whole study.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant decrease of monocytes (38% below controls) recorded in males dosed at 100 mg/kg/day (low dose group) was not dose-related, therefore it was considered to be incidental. In addition, lymphocytes were increased in females receiving 100 mg/kg/day (46% above controls).
Concerning females, those receiving 1000 mg/kg/day (high dose group) showed an increase of leucocytes. Compared with controls, the change was 72% and comprised almost all sub-populations. Due to the low severity and the absence of other related changes, the above finding was considered to be not adverse.
No relevant changes were recorded. The statistically significant increase of prothrombin time (4% above controls) recorded in females dosed at 300 mg/kg/day (Group 3) was not dose-related, therefore it was considered to be incidental. Means of blood clotting time of treated and control animals were comparable.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant fluctuations of some biochemical parameters were recorded between control and animals dosed at 1000mg/kg/day, such as: increase of alanine aminotransferase ( 79%) and sodium (1%) and decrease of potassium (6%) in males, decrease of creatinine (14%) and sodium (1%) and increase of calcium (7%) in females. These changes were insufficient in magnitude to represent an organ/tissue injury, therefore the above findings were considered to be not adverse. Furthermore, the observed changes in liver enzymes were not accompanied with liver weight changes and were without histopathological correlate.
In addition, alanine aminotransferase, urea and creatinine were decreased in females receiving 100 mg/kg/day (23%, 19% and 12% below controls, respectively). Due to the absence of dose-relation, these findings were considered to be unrelated to treatment. Compared with controls, other sporadic changes were recorded, such as: a relevant increase of alanine and aspartate aminotransferases recorded in one female dosed at 1000 mg/kg/day (no. X1080195, 3.2 and 8.0 fold, respectively) and an increase of triglycerides in one other female from the same group (no. X080161, 9.6 fold).
Due to the minimal incidence, the above findings were not attributed to treatment.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No changes were recorded between treated and control animals.

Immunological findings:

no effects observed

Description (incidence and severity):

No relevant differences between control and treated animals were recorded. A single case of increased myeloid cells was observed in female no. X1080073 (100 mg/kg/day); due to the absence of dose-relation and the minimal incidence, this finding was considered to be incidental.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in terminal body weight or absolute and relative organ weights of treated animals of both sexes, when compared to the controls. All organ weight variations between control and treated animals were considered incidental as unrelated to the dose, as all individual values were within the physiological range of variability.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Animals that completed the treatment period andwere killed at termination did not show relevant macroscopic changes that could be considered treatment-related. Sporadic changes included the following: adhesions with heart, trachea, oesophagus, thymus, aorta and ileum, microscopically associated with chronic inflammation and/or fibrosis and/or abscess in one mid-dose female (no. X1080111), indicating a possible misdosing; pale, dark and/or red area or colour of the liver in a single instance for each treated female group (nos. X1080071 - Group 2; X1080115 - Group 3; X1080199 - Group 4) or multiple dark areas
in the glandular stomach of control and treated animals of both sexes. These findings were considered spontaneous and incidental, since also observed in untreated animals under our experimental conditions and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Observations of treated animals at removal from the cage and in an open arena (neurotoxicity assessment) were comparable to controls.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were noted in animals sacrificed at the end of the treatment period.
The sporadic lesions, such as adenocarcinoma in the mammary glands of a low dose female (no. X1010079) correlated to the subcutaneous mass observed during necropsy or hepatocytic necrosis in one control (no. X1080001), low dose (no. X1080071) and high dose (no. X1080155) female, were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age.

Spermatogenic cycle
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Enumeration of ovarian follicles
Differential follicle and corpora lutea counts per ovary did not reveal any relevant differences in all high dose females of parental generation, when compared to the control.

Key result

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 adverse effects observed

Key result

Critical effects observed:

no

Conclusions:

In conclusion, the dosage of 1000 mg/kg/day was considered the NOAEL for general toxicity in this extended one generation reproduction toxicity study.

Executive summary:

In an extended one generation reproduction toxicity study in accordance with OECD TG 443 (adopted on 25 June 2018) thepre- and post-natal effects of MDEA-Esterquat C16-18 and C18 unsatd. on development, as well as a thorough evaluation of systemic toxicity were investigated in male and female rats of the parental generation treated for 10 weeks before pairing and during gestation and lactation period.

In this Iuclid section only the results with the parental generation are considered. The parameters relevant for reproduction are discussed in Iuclid section 7.8.1. Toxicity to reproduction.

The dose levels of 100, 300 and 1000 mg/kg/day were selected for parental animals and were administered orally by gavage. The control group received softened water.

Males were treated for 10 weeks prior to pairing, through the mating period and thereafter until the day before necropsy, for a total of 96-100 days.

Females were treated for 10 weeks prior to pairing, during mating, gestation andpost partumperiods until Day 21post partum, for at least 85 days.

The number of females with live pups on Days 21/22 post partum was: 21 in the control, 22 in the low dose (100 mg/kg/day), 24 in the mid-dose (300 mg/kg/day) and 22 in the high dose (1000 mg/kg/day) groups.

At the daily and weekly clinical observation, no signs considered adverse and no effect in the neurotoxicity assessment were observed.

Body weight, body weight gain and food consumption were unaffected by treatment.

No changes that could be considered adverse were seen in haematology, coagulation and clinical chemistry parameters of treated animals compared to controls.

Exposure to Core 134 (metabolite used to monitor the presence of the test item in plasma) was demonstrated in all treated dams on Day 18post coitumand Day 21post partumat 1 and 4 hours after treatment and in pups of Groups 3 and 4 on Day 21post partum, after approximately 24 hours of treatment of the respective dams. No exposure to Core 134 was detected in pups of Group 2.

No treatment-related anomalies were noted in the oestrous cycle of the treated females, when compared to controls. Copulatory and fertility indices did not show any treatment related differences among treated and control groups. Implantation, pre-natal loss, litter data and sex ratio did not show any changes of toxicological relevance. No significant differences in the anogenital distance were seen between control and treated groups both for male and female pups. No nipples were observed in male pups. Clinical signs and findings at necropsy and organs weight did not reveal any treatment-related or adverse effect.

Sperm analyses performed in all treated males was comparable to controls. Enumeration of ovarian follicles performed in control and high dose females did not show any treatment related effects. No relevant changes were seen in bone marrow evaluation.

No treatment-related changes were noted in animals sacrificed at the end of treatment at organ weight, macroscopic and microscopic examination.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

OECD guideline study, no deviations, GLP

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

One 28 day repeated dose study comparable to OECD Guideline 407 and one sub-chronic toxicity study comparable to OECD Guideline 408 are available for the oral route of administration for the source substance MDEA-Esterquat C16-18 and C18 unsatd.

MDEA-Esterquat C16-18 and C18 unsatd. (10 % a. i. aqueous dispersion) was administered by oral gavage to Charles River CD rats, groups of 25 male and female at dose levels of 1, 10 and 500 mg/kg bw/day for 28 days and to15 rats/sex/dose at dose levels of 1, 10 and 500 mg/kg bw/day for a period of 13-weeks. In both studies, limit doses of 1000 mg/kg bw/day as required by the OECD guidelines were not examined. Furthermore, in both studies there were two control groups, animals received either deionised water (water control group) or pH 2.5 adjusted deionised water (pH-water control group).

In both studies all animals survived until study termination and no significant test substance-related adverse findings were detected or observed for any parameter measured.

In the 28 day study three animals exhibited focal scarring or retinal loss. This finding was not considered to be substance-related and no other test article-related ophthalmoscopic abnormalities were detected. All observations noted were pathologies that would be expected for this group of rats considering their age, sex and strain.

A slight but statistically significant decrease in the MCH index was observed in the male high dose group (500 mg/kg bw/day). However, since no corresponding changes were detected in any other haematologic parameter, this change in the MCH was not considered to be test article related.

 

In the 90 day study a statistically significantly lower mean MCV value was observed in male rates in the water control group and in the 10 and 500 mg/kg bw/day test groups than in the pH-water control group. However, even these lower mean MCV values were within ± 2 standard deviations of laboratory´s historical control mean value for this parameter for rats of this stain, age and sex. Furthermore, they also fall within the ranges of published reference data. Since changes in MCV values were not present in both sexes and since they occurred also in the water control group, they were considered not to be toxicologically significant nor test article-related.

In male rats the blood urea nitrogen values of the 500 mg/kg bw/day group were significantly lower than those of the pH-water control group. These differences were not considered to be test substance-related as there were within the laboratory’s historical control mean values for rats of this strain, age and sex. Individual male and female animals of all groups, including the controls, had elevated values for aspartate aminotransferase (ASAT) activity. These findings in clinical chemistry had no statistical relevance, did not show a dose-response correlation and were without histopathological correlates and therefore they were considered not to be test article-related.

In the 90 day study there were no statistically significant changes in the mean relative ovary/body or ovary/brain weight ratio when compared to both control groups with the exception of a statistically significant decrease in the mean relative ovary/body weight ratio observed in the 500 mg/kg bw/day group when compared to the pH-water control group. The mean body weight of the pH-water control group was the lowest of all female groups in contrast to that of the 500 mg/kg bw/day group, which was the highest. In addition, there were no statistically significant changes in the mean absolute ovary weights. The mean absolute ovary weight of the 500 mg/kg bw/day group falls within the range of the mean absolute ovarian weight for 13-week CD rat studies at the contract laboratory. Therefore, the observed decreased mean ovary/body weight ratio was considered not to be biologically relevant and not test article-related. Individual male and female animals of the 10 mg/kg bw/day group groups, and the control groups, had elevated values for aspartate aminotransferase (ASAT) activity. Also higher alanine aminotransferase (ALAT) activity was seen in individual male and female animals in the 10 mg/kg bw/day group in males and females. These findings in clinical chemistry had no statistical relevance, did not show a dose-response correlation and were without histopathological correlates and therefore they were considered not to be test article-related.

The NOAEL is the highest dose examined, i. e. 500 mg/kg bw/day in both repeated dose toxicity studies. There is no evidence for specific target organ toxicity from both repeated dose toxicity studies.

 

In an extended one generation reproduction toxicity study in accordance with OECD TG 443 (adopted on 25 June 2018) the pre- and post-natal effects of MDEA-Esterquat C16-18 and C18 unsatd.on development, as well as a thorough evaluation of systemic toxicity were investigated in male and female rats of the parental generation treated for 10 weeks before pairing and during gestation and lactation period.

In this Iuclid section only the results with the parental generation are considered. The parameters relevant for reproduction are discussed in Iuclid section 7.8.1. Toxicity to reproduction.

The dose levels of 100, 300 and 1000 mg/kg/day were selected for parental animals and were administered orally by gavage. The control group received softened water.

Males were treated for 10 weeks prior to pairing, through the mating period and thereafter until the day before necropsy, for a total of 96-100 days.

Females were treated for 10 weeks prior to pairing, during mating, gestation and post partum periods until Day 21 post partum, for at least 85 days.

The number of females with live pups on Days 21/22 post partum was: 21 in the control, 22 in the low dose (100 mg/kg/day), 24 in the mid-dose (300 mg/kg/day) and 22 in the high dose (1000 mg/kg/day) groups.

At the daily and weekly clinical observation, no signs considered adverse and no effect in the neurotoxicity assessment were observed.

Body weight, body weight gain and food consumption were unaffected by treatment.

No changes that could be considered adverse were seen in haematology, coagulation and clinical chemistry parameters of treated animals compared to controls.

Exposure to Core 134 (metabolite used to monitor the presence of the test item in plasma) was demonstrated in all treated dams on Day 18post coitumand Day 21post partumat 1 and 4 hours after treatment and in pups of Groups 3 and 4 on Day 21post partum, after approximately 24 hours of treatment of the respective dams. No exposure to Core 134 was detected in pups of Group 2.

No treatment-related anomalies were noted in the oestrous cycle of the treated females, when compared to controls. Copulatory and fertility indices did not show any treatment related differences among treated and control groups. Implantation, pre-natal loss, litter data and sex ratio did not show any changes of toxicological relevance. No significant differences in the anogenital distance were seen between control and treated groups both for male and female pups. No nipples were observed in male pups. Clinical signs and findings at necropsy and organs weight did not reveal any treatment-related or adverse effect.

Sperm analyses performed in all treated males was comparable to controls. Enumeration of ovarian follicles performed in control and high dose females did not show any treatment related effects. No relevant changes were seen in bone marrow evaluation.

No treatment-related changes were noted in animals sacrificed at the end of treatment at organ weight, macroscopic and microscopic examination.

The NOAEL is the highest dose examined, i. e. 1000 mg/kg bw/day.

Conclusion

No adverse effects were observed in any of the available studies. The 90 d repeated dose toxicity study was not conducted up to the limit dose; the highest tested dose level was 500 mg/kg bw/d in this study. However, the EOGRTS, which was conducted up to the limit dose of 1000 mg/kg bw/d, contains a thorough examination of systemic toxicity covering all relevant parameters of a subchronic repeated dose toxicity study. Therefore, the NOAEL of 1000 mg/kg bw/d obtained in the EOGRTS is considered as the key value for hazard assessment.

 

There are no data gaps for the endpoint repeated dose toxicity. No human data are available. However, there is no reason to believe that these results from rat would not be applicable to humans.

Justification for classification or non-classification

In conclusion, results of both studies indicate that MDEA-Esterquat C16-18 and C18 unsatd.  does not need to be classified for repeated dose toxicity according to GHS Regulation EC No 1272/2008 and therefore labelling is not necessary.