Tributylethylammonium ethyl sulphate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 2016 - 13 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 2016 - 13 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

March 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

Remarks:

(

Qualifier:

according to guideline

Guideline:

other: other guidance as listed under Principles of method if other than guideline

Principles of method if other than guideline:

In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 1995);
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 10-11 weeks.
- Weight at study initiation: 267-302 gr (males) or 190-225 gr (females)
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 15 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 23 November 2016 to 13 Janruary 2017

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1%

Details on oral exposure:

Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No correction was made for the purity/composition of the test item. In order to obtain homogeneity, the formulations were heated up to a maximum temperature of 64,8ºC for a maximum of 31 minutes. The formulations were cooled down to a
temperature of maximally 40ºC prior to dosing.
Storage conditions of formulations: At room temperature.
Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice.

The following measurements in formulations were performed for the cationic and anionic part of the test item using LC-MS/MS:
- Cationic part: Samples of formulations were analyzed for accuracy of preparation (all concentrations) and homogeneity (highest and lowest concentration).
- Anionic part: Samples of formulations were analyzed for accuracy of preparation (all concentrations), and homogeneity and stability in vehicle over 5 hours at room temperature (highest and lowest concentration).
Note: Since the in-life phase of the study was completed, separate dose preparationsnwere prepared according to the same protocol as during in-life.

The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to and including the day before termination. Females were exposed for 42-45 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation up to and including the day before scheduled necropsy.
The female that failed to deliver healthy offspring was treated for 42 days.
Routinely, females that are littering are left undisturbed. In this study, one control female was left out from treatment for one day as she was littering at the moment of dosing.

Frequency of treatment:

Once daily, 7 d/w

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:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on the results of a 10-day dose range finding study, the dose levels for this combined 28-day oral gavage study with the reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg.

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.

Positive control:

No.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals, at least 1 hour (± 30 min) after dosing (on the peak period of anticipated effects after treatment). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT:
Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
Yes. Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
Yes. (average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m..
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m..
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (from lactation Day 4 onwards). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: According to test guidelines

Deviations:
For one litter (100 mg/kg bw/d) no pup body weights were recorded on PND 4.
The outcome of the pup observations for 2 litters of the control group, 2 litters of the 100 mg/kg/d group and one litter of the 300 mg/kg bw/d group on PND 4, one litter of the 1000 mg/kg bw/d group on PND 2 and PND 4, and one liter of the 1000 mg/kg bw/day group on PND 4 was not recorded. For two pups of one litter of the 300 mg/kg bw/d group no last litter check and macroscopic examination was performed.
The study integrity was not adversely affected by these deviations.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

All males and selected femalesn were fasted overnight (with a maximum of 24.9 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy were deeply anaesthetised and subsequently exsanguinated.

- Selected 5 animals/sex/group: According to test guidelines

- All remaining animals, one male that had failed to sire (100 mg/kg bw/d), one female (100 mg/kg bw/d) which had failed to deliver: According to test guidelines

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines

- All remaining males:
Epididymides and Testes

HISTOPATHOLOGY: Yes
According to test guidelines

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation seen after dosing among females of the 300 and 1000 mg/kg bw/day dose group on one or two days during week 5 of the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
Incidental findings that were noted in females at dose groups 100, 300 and 1000 mg/kg bw/day included alopecia, scales, scabbing, and exophthalmos of the eye. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be signs of toxicological relevance. No clinical signs were noted in males.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.
One male at 300 mg/kg bw/day died due to complications during blood sampling on the day of scheduled necropsy (Day 30).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significantly lower body weight gains recorded for males at 1000 mg/kg bw/day compared to the concurrent control group during the mating period was not considered treatment-related as a similar trend (not reaching statistical significance) was observed for males at 100 mg/kg bw/day, but not 300 mg/kg bw/day. Neither the lower body weight gain noted in females at 1000 mg/kg bw/day at the end of the post-coitum period (Day 20) was considered to be related to treatment as it remained within the normal range of biological variation and normal body gains were seen during lactation again.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Any statistically significant changes were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution, differences compared to the concurrent control group were slight and all values remained within the range considered normal for rats of this age and strain.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Any statistically significant changes were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution, differences compared to the concurrent control group were slight and all values remained within the range considered normal for rats of this age and strain.

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):

Any statistically significant changes were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The lower mean values of total protein, albumin and chloride noted in males at 1000 mg/kg bw/day as compared to the concurrent control group were not considered treatment-related as differences compared to the concurrent control group were slight and all values remained within the range considered normal for rats of this age and strain.
Any remaining statistically significant changes were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution and remained within the normal range of biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significantly higher absolute weight of the heart in females treated with 300 mg/kg bw/day occurred in the absence of a dose-related trend. As such, this variation was not considered to be related to treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
One male at 1000 mg/kg bw/day was noted with:
- A papillary liver process that was thickened (left side), hardened and with many darkred foci. This finding corresponded with hemorrhages with marked necrosis at the microscopic level.
- A hard nodule (7x5 mm) on the epididymal adipose tissue (right side) in the abdominal cavity. At the microscopic level marked encapsulated necrosis of the fat tissue with inflammation was noted.
- An enlarged thyroid gland (both sides) without any corresponding microscopic changes.
At the isolated incidence, this finding was not considered to be related to treatment.

The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend.

Histopathological findings: non-neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

Parental generation

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicity was observed up to and including the highest dose level tested.

Critical effects observed:

no

Analysis of dose preparations:

Cationic part of test item:

In the control group formulation, no test substance was detected. The concentrations analysed in the formulations of groups exposed to 100, 300 and 1000 mg/kg bw/day were in agreement with target concentrations (i.e. mean accuracies 100.4%, 103.3% and 101.1% respectively).

The formulations of the low and high dose group were homogeneous (i.e. coefficient of variation 2.1% and 2.0%, respectively).

Stability for the cationic part of the test item was already was already demonstrated as part of the analytical Method Development and Validation. Stability for at least 5 hours at room temperature and

8 days in the refrigerator is confirmed over the concentration range 1 to 200 mg/mL (Project 513826).

Anionic part of the test item:

In the control group formulation, no test substance was detected. The concentrations analysed in the formulations of groups exposed to 100, 300 and 1000 mg/kg bw/day were in agreement with target concentrations (i.e. mean accuracies 95.5%, 94.0% and 94.1% respectively).

The formulations of the low and high dose group were homogeneous (i.e. coefficient of variation 3.9% and 5.2%, respectively). Recovery after 6 hours was found to be 99.7% and 108.3% for the high (approx. 200 mg/g) and low (approx. 1 mg/g) recovery samples, respectively. Based on this, the formulations were found to be stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Conclusions:

In an oral OECD422/421 screening study with rats, the NOAEL was determined to be ≥1000 mg/kg bw/day, based on the absence of adverse effects up to and including 1000 mg/kg bw/day

Executive summary:

A combined 28d repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. TBEAES was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42 -45 days). The female which failed to deliver healthy offspring was treated for 42 days.

Formulation analysis for the cationic and anionic part of TBEAES DRY showed that the formulations were prepared accurately and homogenously, and were stable for at least 5 hours at room temperature.

No mortality occurred during the study period that was considered to be related to treatment with the test item. No clinical signs of toxicity were noted during the observation period. No toxicologically relevant changes in body weights and body weight gain were noted. Food consumption, functional observation parameters, haematological parameters, clinical biochemistry parameters and organ weights were not considered to be affected by treatment. One male at 1000 mg/kg bw/day was noted with a pappilary liver proces, a hard nodule on the epididymal adipose tissue in the abdominal cavity and an enlarged thyroid gland. This finding was considered to be incidental and not treatment related. There were no test item-related microscopic observations.

Based on the absence of adverse effects up to and including 1000 mg/kg bw/day, a No Observed Adverse Effect Level (NOAEL) for TBEAES DRY of ≥ 1000 mg/kg bw/day was established.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Remarks:

oral combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 2016 - 13 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: Organisation of Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

March 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: other guidance as listed under Principles of method if other than guideline

Deviations:

no

Principles of method if other than guideline:

In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 1995);
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 10-11 weeks.
- Weight at study initiation: 267-302 gr (males) or 190-225 gr (females)
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage with out cage-enrichment, bedding material, food and water.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest,Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 15 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 November 2016 to 13 Janruary 2017

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1%

Details on exposure:

Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No correction was made for the purity/composition of the test item. In order to obtain homogeneity, the formulations were heated up to a maximum temperature of 64,8ºC for a maximum of 31 minutes. The formulations were cooled down to a temperature of maximally 40ºC prior to dosing.

Storage conditions of formulations: At room temperature.

Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples (0.5 mL) were taken and weighed. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. The following measurements in formulations were performed for the cationic and anionic part of the test item using LC-MS/MS:
- Cationic part: Samples of formulations were analyzed for accuracy of preparation (all concentrations) and homogeneity (highest and lowest concentration).
- Anionic part: Samples of formulations were analyzed for accuracy of preparation (all concentrations), and homogeneity and stability in vehicle over 5 hours at room temperature (highest and lowest concentration). Note: Since the in-life phase of the study was completed, separate dose preparations were prepared according to the same protocol as during in-life.

Analysis of dose preparations:
Cationic part of test item:
In the control group formulation, no test substance was detected. The concentrations analysed in the formulations of groups exposed to 100, 300 and 1000 mg/kg bw/day were in agreement with target concentrations (i.e. mean accuracies 100.4%, 103.3% and 101.1% respectively). The formulations of the low and high dose group were homogeneous (i.e. coefficient of variation 2.1% and 2.0%, respectively).
Stability for the cationic part of the test item was already was already demonstrated as part of the analytical Method Development and Validation. Stability for at least 5 hours at room temperature and 8 days in the refrigerator is confirmed over the concentration range 1 to 200 mg/mL (Project 513826).

Anionic part of the test item:
In the control group formulation, no test substance was detected. The concentrations analysed in the formulations of groups exposed to 100, 300 and 1000 mg/kg bw/day were in agreement with target concentrations (i.e. mean accuracies 95.5%, 94.0% and 94.1% respectively). The formulations of the low and high dose group were homogeneous (i.e. coefficient of variation 3.9% and 5.2%, respectively). Recovery after 6 hours was found to be 99.7% and 108.3% for the high (approx. 200 mg/g) and low (approx. 1 mg/g) recovery samples, respectively. Based on this, the formulations were found to be stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Age at mating of the mated animals in the study: Approximately 13 weeks
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42-45 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Routinely, females that are littering are left undisturbed. In this study, one control female was left out from treatment for one day as she was littering at the
moment of dosing.
Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

Once daily for 7 d/w.

Duration of test:

Males: 29 days
Females: 42-45 days. The female that failed to deliver healthy offspring was treated for 42 days.

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:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on the results of a 10-day dose range finding study, the dose levels for this combined 28-day oral gavage study with the reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg.
The peak effect of occurrence of clinical signs occurred at 1 hours after dosing. This time point was taken into account to set a time range within which clinical observations and functional observation tests had to be conducted after dosing in the main study.

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.

Identification of pups:
On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.

Maternal examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals, at least 1 hour (± 30 min) after dosing (on the peak period of anticipated effects after treatment). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT
- Time schedule for examinations:Females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION
Weekly. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on
Days 1 and 4 of lactation.

FOOD EFFICIENCY
(Average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION : No
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

GENERAL REPRODUCTION DATA
- Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.
- Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/group
- Parameters checked were: According to test guidelines

URINALYSIS:
No

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: The selected females were tested during lactation (all before blood sampling). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Dose groups that were examined: all (5 animals/group)
- Battery of functions tested: According to test guidelines

GROSS PATHOLOGY
All animals were fasted overnight (with a maximum of 24.9 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy were deeply anaesthetised and subsequently exsanguinated.
The number of former implantation sites and corpora lutea were recorded for all paired females.
- Selected 5 animals/group: According to test guidelines
- All remaining animals: one female (100 mg/kg bw/day) which had failed to deliver: According to test guidelines

ORGAN WEIGHTS
- Selected 5 animals/group: According to test guidelines

HISTOPATHOLOGY
According to test guidelines.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.
- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

SACRIFICE
Pups surviving to planned termination were killed by decapitation between Days 5-6 of lactation.

GROSS EXAMINATION OF DEAD PUPS: Yes
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination. Pups 1 and 2 of a litter in the 300 mg/kg bw/day group found dead during the weekend were fixed in an identified container containing 70% ethanol as they were not necropsied on the same day.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one ttest) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.

Indices:

For each group, the following calculations were performed:
- precoital time: Number of days between initiation of cohabitation and confirmation of mating
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring indices:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss: Number of dead pups before planned necropsy/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups before planned necropsy / Number of pups born alive x 100

Group mean values were calculated from individual litter values.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation seen after dosing among females of the 300 and 1000 mg/kg bw/day dose group on one or two days during week 5 of the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
Incidental findings that were noted in females at dose groups 100, 300 and 1000 mg/kg bw/day included alopecia, scales, scabbing, and exophthalmos of the eye. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be signs of toxicological relevance.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The lower body weight gain noted in females at 1000 mg/kg bw/day at the end of the post-coitum period (Day 20) was not considered to be related to treatment as it remained within the normal range of biological variation and normal body gains were seen during lactation again.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Any statistically significant changes were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution, differences compared to the concurrent control group were slight and all values remained within the range considered normal for rats of this age and strain.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Any statistically significant changes were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution, differences compared to the concurrent control group were slight and all values remained within the range considered normal for rats of this age and strain.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Any statistically significant changes were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution and remained within the normal range of biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significantly higher absolute weight of the heart in females treated with 300 mg/kg occurred in the absence of a dose-related trend. As such, this variation was not considered to be related to treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Number of abortions:

not examined

Pre- and post-implantation loss:

not examined

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Gestation index was not affected by treatment, all pregnant females delivered pups.

Early or late resorptions:

not examined

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Duration of gestation was not affected by treatment. All pregnant females delivered live pups after 20-23 days of gestation.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

A single female at 100 mg/kg was not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was not considered to be related to treatment.

Details on maternal toxic effects:

No maternal toxicity was observed up to 1000 mg/kg.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: Absence of adverse effects at highest dose level tested.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were not considered to be affected by treatment.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment.
Two pups ofa litter in the 300 mg/kg bw/day dose group, were found dead and partially cannibalized at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

The number of alive pups before planned necropsy compared to the number of pups born alive was not affected by treatment. One pup of a control litter, one pup at 100 mg/kg bw/day, 6 pups at 300 mg/kg bw/day, and one pup at 1000 mg/kg bw/day were found missing. Four out of the six pups missing at 300 mg/kg came from the same litter. However, as the remaining 11 pups in this litter developed normally and survived until scheduled necropsy on PND 5, no toxicological relevance was attached to this finding. Pups missing were most likely cannibalised.

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

not examined

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.

Details on embryotoxic / teratogenic effects:
There was no evidence of teratogenic effects based on the absence of relevant clinical signs and external macroscopic findings.

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: Absence of adverse effects at the highest dose level tested.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In an oral OECD 422/421 screening study with rats, the parental and developmental NOAEL for TBEAES DRY were determined to be ≥1000 mg/kg bw/day, based on the absence of adverse effects up to and including 1000 mg/kg bw/day

Executive summary:

A combined 28d repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and in accordance with GLP principles. TBEAES was administered by daily oral gavage to male and female rats at dose levels of 100, 300 and 1000 mg/kg bw/ day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42 -45 days). The female which failed to deliver healthy offspring was treated for 42 days. Formulation analysis for the cationic and anionic part of TBEAES DRY showed that the formulations were prepared accurately and homogenously, and were stable for at least 5 hours at room temperature.

No parental toxicity was observed up to and including 1000 mg/kg. No treatment-related changes were noted in any of the parental parameters investigated in this study (i.e. mortality, clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

No developemtal toxicity was observed up and including 1000 mg/kg bw/day. No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

Based on the absence of adverse effects o, a parental and developmental No Observed Adverse Effect Level (NOAEL) for TBEAES DRY of ≥ 1000 mg/kg bw/ day was established.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

March 1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000)

Version / remarks:

July 2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: other guidance as listed under Principles of method if other than guideline

Principles of method if other than guideline:

In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 1995);
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tributylethylammonium ethyl sulphate

EC Number:

268-329-4

EC Name:

Tributylethylammonium ethyl sulphate

Cas Number:

68052-51-7

Molecular formula:

C14H32N . C2H5O4S (or C16H37NO4S)

IUPAC Name:

tributyl(ethyl)azanium ethyl sulfate

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

Test material form:

solid

Details on test material:

- Physical appearance: off white solid
- Storage conditions: at room temperature

Test animals

Species:

rat

Strain:

other: Crl:WI(Han).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 10-11 weeks.
- Weight at study initiation: 267-302 gr (males) or 190-225 gr (females)
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage with out cage-enrichment, bedding material, food and water.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 15 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 November 2016 to 13 Janruary 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1%

Details on exposure:

Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No correction was made for the purity/composition of the test item. In order to obtain homogeneity, the formulations were heated up to a maximum temperature of 64,8ºC for a maximum of 31 minutes. The formulations were cooled down to a temperature of maximally 40ºC prior to dosing.
Storage conditions of formulations: At room temperature.
Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples (0.5 mL) were taken and weighed. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. The following measurements in formulations were performed for the cationic and anionic part of the test item using LC-MS/MS:
- Cationic part: Samples of formulations were analyzed for accuracy of preparation (all concentrations) and homogeneity (highest and lowest concentration).
- Anionic part: Samples of formulations were analyzed for accuracy of preparation (all concentrations), and homogeneity and stability in vehicle over 5 hours at room temperature (highest and lowest co
ncentration). Note: Since the in-life phase of the study was completed, separate dose preparations were prepared according to the same protocol as during in-life.

Analysis of dose preparations:
Cationic part of test item:
In the control group formulation, no test substance was detected. The concentrations analysed in the formulations of groups exposed to 100, 300 and 1000 mg/kg bw/day were in agreement with target concentrations (i.e. mean accuracies 100.4%, 103.3% and 101.1% respectively). The formulations of the low and high dose group were homogeneous (i.e. coefficient of variation 2.1% and 2.0%, respectively).
Stability for the cationic part of the test item was already was already demonstrated as part of the analytical Method Development and Validation. Stability for at least 5 hours at room temperature and
8 days in the refrigerator is confirmed over the concentration range 1 to 200 mg/mL (Project 513826).
Anionic part of the test item:
In the control group formulation, no test substance was detected. The concentrations analysed in the formulations of groups exposed to 100, 300 and 1000 mg/kg bw/day were in agreement with target concentrations (i.e. mean accuracies 95.5%, 94.0% and 94.1% respectively). The formulations of the low and high dose group were homogeneous (i.e. coefficient of variation 3.9% and 5.2%, respectively). Recovery after 6 hours was found to be 99.7% and 108.3% for the high (approx. 200 mg/g) and low (approx. 1 mg/g) recovery samples, respectively. Based on this, the formulations were found to be stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42-45 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Routinely, females that are littering are left undisturbed. In this study, one control female was left out from treatment for one day as she was littering at the moment of dosing.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

Once daily for 7 d/w.

Details on study schedule:

- Age at mating of the mated animals in the study: Approximately 13 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on the results of a 10-day dose range finding study, the dose levels for this combined 28-day oral gavage study with the reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg.
The peak effect of occurrence of clinical signs occurred at 1 hours after dosing. This time point was taken into account to set a time range within which clinical observations and functional observation tests had to be conducted after dosing in the main study.

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.

Identification of pups:
On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals, at least 1 hour (± 30 min) after dosing (on the peak period of anticipated effects after treatment). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT
- Time schedule for examinations:Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION
Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY
(Average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION
No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION
No

HAEMATOLOGY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m..
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m..
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked in table were: According to test guidelines

URINALYSIS
No

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: According to test guidelines

Oestrous cyclicity (parental animals):

Not determined.

Sperm parameters (parental animals):

Slides of the testes were prepared for histopathological staging of spermatogenesis (5 males of the control and high dose group).

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.

Deviations:
- For one litter in the 100 mg/kg bw/day group no pup body weights were recorded on PND 4.
- The outcome of the pup observations for two control litters, two litters in the 100 mg/kg bw/day group and one litter in the 300 mg/kg bw/day group on PND 4, one litter in the 1000 mg/kg bw/day group on PND 2 and PND 4, and one litter of the 1000 mg/kg bw/day group on PND 4 was not recorded. For pups 13 and 14 of one 300 mg/kg bw/day litter no last litter check and macroscopic examination was performed.
Sufficient data was available from the other litters for adequate toxicological evaluation.

Postmortem examinations (parental animals):

GROSS PATHOLOGY
All animals were fasted overnight (with a maximum of 24.9 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy were deeply anaesthetised and subsequently exsanguinated.

The number of former implantation sites and corpora lutea were recorded for all paired females.

- Selected 5 animals/sex/group: According to test guidelines

- All remaining animals one male that had failed to sire (100 mg/kg bw/day), one female (100 mg/kg bw/day) which had failed to deliver: According to test guidelines

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines (+ deviations)

- All remaining males:
Epididymides and Testes

HISTOPATHOLOGY
According to test guidelines.

Postmortem examinations (offspring):

SACRIFICE
Pups surviving to planned termination were killed by decapitation between Days 5-6 of lactation.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.
Pups 1 and 2 of a litter in the 300 mg/kg bw/day group found dead during the weekend were fixed in an identified container containing 70% ethanol as they were not necropsied on the same day.

HISTOPATHOLOGY / ORGAN WEIGTHS
No.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.

Reproductive indices:

For each group, the following calculations were performed:
- precoital time: Number of days between initiation of cohabitation and confirmation of mating
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss: Number of dead pups before planned necropsy/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups before planned necropsy / Number of pups born alive x 100

Group mean values were calculated from individual litter values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation seen after dosing among females of the 300 and 1000 mg/kg bw/day dose group on one or two days during week 5 of the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
Incidental findings that were noted in females at dose groups 100, 300 and 1000 mg/kg bw/day included alopecia, scales, scabbing, and exophthalmos of the eye. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be signs of toxicological relevance. No clinical signs were noted in males.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.
One male at 300 mg/kg bw/day died due to complications during blood sampling on the day of scheduled necropsy (Day 30).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significantly lower body weight gains recorded for males at 1000 mg/kg bw/day compared to the concurrent control group during the mating period was not considered treatment-related as a similar trend (not reaching statistical significance) was observed for males at 100 mg/kg bw/day, but not 300 mg/kg bw/day. Neither the lower body weight gain noted in females at 1000 mg/kg bw/day at the end of the post-coitum period (Day 20) was considered to be related to treatment as it remained within the normal range of biological variation and normal body gains were seen during lactation again.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Any statistically significant changes were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution, differences compared to the concurrent control group were slight and all values remained within the range considered normal for rats of this age and strain.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Any statistically significant changes were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution, differences compared to the concurrent control group were slight and all values remained within the range considered normal for rats of this age and strain.

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):

Any statistically significant changes were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The lower mean values of total protein, albumin and chloride noted in males at 1000 mg/kg bw/day as compared to the concurrent control group were not considered treatment-related as differences compared to the concurrent control group were slight and all values remained within the range considered normal for rats of this age and strain.
Any remaining statistically significant changes were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution and remained within the normal range of biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There was 1/10 couples of the 100 mg/kg/day group with no offspring. Histopathology did not reveal any changes in the reproductive organs that could explain this finding.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and spermatogenic staging profiles were normal for all males examined.

Effect levels (P0)

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 toxicity was observed up to and including the highest dose level tested.

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Two pups of a litter in the 300 mg/kg bw/day group were found dead and partially cannibalized at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
One pup of a control litter, one pup at 100 mg/kg bw/day, 6 pups at 300 mg/kg bw/day, and one pup at 1000 mg/kg bw/day were found missing. Four out of the six pups missing at 300 mg/kg came from the same litter. However, as the remaining 11 pups in this litter developed normally and survived until scheduled necropsy on PND 5, no toxicological relevance was attached to this finding. Pups missing were most likely cannibalised.

Body weight and weight changes:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental macroscopic findings of pups that were found dead included cannibalism. The nature and incidence of the macroscopic findings noted incidentally among surviving pups remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Histopathological findings:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicity was observed up to and including the highest dose level tested.

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In an oral OECD 422/421 screening study, the parental NOAEL and the NOAEL for reproduction for TBEAES DRY were derived to be ≥1000 mg/kg bw/day, based on the absence of adverse effects up to and including 1000 mg/kg bw/day.

Executive summary:

A combined 28d repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. TBEAES was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42 -45 days). The female which failed to deliver healthy offspring was treated for 42 days.

Formulation analysis for the cationic and anionic part of TBEAES DRY showed that the formulations were prepared accurately and homogenously, and were stable for at least 5 hours at room temperature.

No parental toxicity was observed up to and including 1000 mg/kg. No treatment-related changes were noted in any of the parental parameters investigated in this study (i.e. mortality, clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

No reproduction toxicity was observed up to and including 1000 mg/kg bw/day. No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, and numbers of corpora lutea and implantations, spermatogenic profiling, and histopathological examination of reproductive organs).

 

Based on the absence of adverse effects up to and including 1000 mg/kg bw/day, a parental No Observed Adverse Effect Level (NOAEL) for TBEAES DRY of ≥1000 mg/kg bw/day was established. The NOAEL for reproduction was established at ≥1000 mg/kg bw/day.