Ethyltriphenylphosphonium acetate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October 2017 - 07 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October 2017 - 07 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han™:RccHan™:WIST

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males: approx. 11 weeks, females: approx. 14 weeks
- Weight at study initiation: males: 289 to 356g; females: 192 to 246g
- Fasting period before study: no
- Housing: groups of three (non-recovery) or five (recovery) in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
During the pairing phase, non-recovery animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the non-recovery males were returned to their original cages. Mated non-recovery females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) ad libitum
- Water (e.g. ad libitum): mains drinking water ad libitum
- Acclimation period: least two weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 15/h
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Vehicle:

other: 0.5% Tween/1% Carboxy Methylcellulose

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared fortnightly and stored at approximately 4 ºC in the dark

- VEHICLE
- Concentration in vehicle: 2, 5, 15 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test item formulations were taken on three occasions and analyzed for concentration. The results indicate that the prepared formulations were within 97-103 % of the nominal concentration. Results showed the formulations to be stable for at least twenty-one days when stored refrigerated. Formulations were therefore prepared fortnightly and stored at approximately 4 ºC in the dark.

Analysis was performed by HPLC + UV detection
HPLC: Agilent Technologies 1200 with autosampler
Mobile phase: Methanol + 0.1% Formic acid : 100 mM Ammonium Formate + 0.1% Formic acid (85:15, v/v)
Column: Synergi Polar RP 4µ

Duration of treatment / exposure:

non-recovery animals: females: up to 10 weeks (14 d pre-mating, max. 14 d mating phase, throughout pregnancy, 13 d post partum)
males: total 44 d
recovery animals: dosed according to dose group continuously up to the point of sacrifice of non-recovery males at which time treatment was discontinued + additional 28 days without treatment

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12 (main groups), 5 (recovery groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were chosen in collaboration with the Sponsor and were based on the results of previous toxicity work including a Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat (Envigo Study Number: MV50QQ). In this study one female treated with 500 mg/kg bw/day was found dead on Day 1 after dosing and two females from this treatment group were sacrificed in extremis due to excessive clinical signs on Day 1. The remaining animals treated with 500 mg/kg bw/day were sacrificed for animal welfare reasons on Day 1. One male treated with 150 mg/kg bw/day was found dead on Day 11 of treatment and the remaining animals at this level were sacrificed on the same day due to excessive dose level. Reductions in body weight gain and food consumption were evident in either sex at 150 mg/kg bw/day and reductions in body weight gain were evident in either sex treated with 100 mg/kg bw/day. At necropsy, macroscopic stomach findings were evident at 500 mg/kg bw/day and in the male that was found dead at 150 mg/kg bw/day. No effects were evident in animals of either sex treated with 50 mg/kg bw/day.

- Rationale for selecting satellite groups: to assess the ability of the animals to recover from any toxicity over twenty-eight days following the withdrawal of treatment
- Post-exposure recovery period in satellite groups: 28 d

Positive control:

not applicable

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of treatment and at approximately weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioral toxicity (non-recovery mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum). Functional performance tests were also performed on five selected non-recovery males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
Behavioural assessment: Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 (prior to dosing) for non-recovery animals and then weekly for non-recovery males until termination and weekly for non-recovery females until pairing. During pairing phase non-recovery females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4, 7 and 14 post partum. Recovery animals were weighed on Day 1 (prior to dosing) and then approximately weekly until termination. Body weights were also recorded at terminal sacrifice.

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

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: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily by visual inspection of water bottles for any overt changes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 43 for males and Day 13 post partum for females; recovery group animals after the twenty-eight day treatment-free period (Day 71)
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: No
- How many animals: 5
- Parameters: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices (mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), Total leukocyte count (WBC), Differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT), Reticulocyte count (Retic)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 43 for males and Day 13 post partum for females; recovery group animals after the twenty-eight day treatment-free period (Day 71)
- Animals fasted: No
- How many animals: 5
- Parameters: Urea, Inorganic phosphorus (P), Glucose, Aspartate aminotransferase (ASAT), Total protein (Tot.Prot.), Alanine aminotransferase (ALAT), Albumin, Alkaline phosphatase (AP), Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat), Sodium (Na+), Total cholesterol (Chol), Potassium (K+), Total bilirubin (Bili), Chloride (Cl-), Bile acids, Calcium (Ca++)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: non-recovery mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum; five selected non-recovery males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No.

Thyroid Hormone Analysis (adult males)
- Time schedule: at termination
- T4
- serum from non-recovery adult males

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Examination of offspring was restricted to a macroscopic external examination except where abnormalities were observed; then an additional internal examination was performed.

HISTOPATHOLOGY: Yes (see table)

Other examinations:

Thyroid Hormone Analysis (offspring) (detailed in Iuclid chapter 7.8)
Reproductive parameters (detailed in Iuclid chapter 7.8)

Statistics:

see "Any other information on materials and methods"

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The majority of animals of either sex treated with 75 mg/kg bw/day showed increased salivation from Day 10 to Day 42 (males) and from Day 14 to Day 57 (females). Incidences of noisy respiration were also evident in three males and four females from this treatment group between Days 15 and 44 (males) and Day 31 and Day 63 (females). One female treated with 25 mg/kg bw/day showed increased salivation on Days 45 and 50 and another female from this treatment group had noisy respiration on Day 20. At 10 mg/kg bw/day, two females had noisy respiration on Day 20. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and are considered not to represent true systemic toxicity.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no treatment-related deaths.
One female treated with 75 mg/kg bw/day was sacrificed in extremis on Day 14, due to the severity of clinical observations. The animal was noted to have mis-aligned posture with the head tilted to the left from Day 11 and exophthalmos in the left eye from Day 13. By Day 14 the head tilt had become more severe with the head being held in an almost upside down position, loss of righting reflex and increased respiratory rate were also observed. Macroscopic necropsy revealed no abnormalities, although minimal cortical hypertrophy in the adrenal gland was observed at histopathology, indicating a minor stress-related change. The cause of the clinical observations could not conclusively be determined; however, findings of this nature are occasionally seen in laboratory maintained rats and are often attributed to inflammation or infection of the middle or inner ear, which can affect balance. The death was not considered to be attributed to the administration of the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males treated with 75 mg/kg bw/day showed a slight reduction in body weight gain during the first four weeks of treatment, although, statistical significance was not achieved. Improvement was evident thereafter, with body weight gains during Week 6 showing statistically significantly higher values when compared to controls. Body weight gain for recovery animals during the treatment-free period was comparable to controls.
No adverse effect in body weight development was evident in treated females during maturation. Females treated with 75 mg/kg bw/day showed a reduction in body weight gain during the first two weeks of gestation, with cumulative body weight gain between Days 0-14 of gestation being statistically significantly lower than controls. Recovery was evident during the final week of gestation and no adverse effects were seen during lactation.
Body weights and body weight gains were unaffected by treatment in both sexes at 25 and 10 mg/kg bw/day throughout treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No effect on food consumption was evident in treated males.
No effect on food consumption (maturation only) was evident in treated females during maturation, gestation or lactation.

Food efficiency:

no effects observed

Description (incidence and severity):

No effect on food conversion efficiency was evident in treated males.
No effect on food conversion efficiency (maturation only) was evident in treated females during maturation, gestation or lactation.

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

no effects observed

Description (incidence and severity):

Daily visual assessment of water consumption did not reveal any significant intergroup differences.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of hematology parameters did not indicate any obvious effect of treatment for either sex at 10, 25 or 75 mg/kg bw/day.
Mean corpuscular hemoglobin concentration for non-recovery males treated at 75 mg/kg bw/day and all non-recovery treated females were statistically significantly lower than controls (p<0.05 - p<0.01). Reticulocytes for all non-recovery treated males and non-recovery females treated with 75 and 25 mg/kg bw/day were statistically significantly higher (p<0.05 - p<0.01) than controls. All individual values were within historical control ranges and in the absence of any histopathological correlates or in the case of male reticulocyte count, no true dose related response, the intergroup differences were considered not to be of toxicological significance.
Eosinophils and platelet values for males previously treated with 75 mg/kg bw/day were statistically significantly increased (p<0.05) and reduced (p<0.05), respectively, at the end of the treatment free period. The majority of individual values for these parameters were within historical control ranges and in the absence of a similar effect in non-recovery animals at the end of the treatment period, the intergroup differences were considered not to be of toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of blood chemistry parameters did not indicate any obvious effect of treatment for either sex at 10, 25 or 75 mg/kg bw/day.
Non-recovery males treated with 75 mg/kg bw/day showed statistically significant reductions in albumin/globulin ratio (p<0.05), chloride concentration (p<0.05), bilirubin (p<0.01) and alkaline phosphatase (p<0.01). Inorganic phosphorus was statistically significantly increased (p<0.01) for these males and bilirubin continued to be significantly reduced (p<0.05) in recovery males that were previously treated with 75 mg/kg bw/day, following the treatment free period. The majority of individual values for these parameters were within historical control ranges and in the absence of any histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Females previously treated with 75 mg/kg bw/day showed a statistically significantly reduction (p<0.05) in potassium concentration at the end of the treatment free period. All of the individual values were within the historical control range and in the absence of a similar effect in non-recovery females at the end of the treatment period, the intergroup difference was considered not to be of toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse changes in the behavioral parameters at 10, 25 or 75 mg/kg bw/day.
Two females treated with 75 mg/kg bw/day showed noisy respiration during assessments on Day 4 and/or Day 12 of lactation. These observations correlated to the daily clinical signs evident at this dose level and were due to the oral administration of an unpalatable or slightly irritant test item formulation rather than evidence of true systemic toxicity.
Mis-aligned posture as evident in the daily clinical observations was noted during the second week of assessments for the female treated with 75 mg/kg bw/day that was sacrificed in extremis. One control male had pilo-erection during the final week of assessment. In the absence of treatment this was considered incidental.

There were no changes in functional performance at 10, 25 or 75 mg/kg bw/day.

There were no inter-group differences in sensory reactivity scores at 10, 25 or 75 mg/kg bw/day.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related effects detected in the organ weights measured at 10, 25 or 75 mg/kg bw/day.
In recovery females previously treated with 75 mg/kg bw/day, both absolute and terminal body weight relative spleen weights were statistically significantly higher (p<0.01) than controls. All of the individual values were within historical control ranges and in the absence of a similar effect in non-recovery females at the end of the treatment period or any histopathological correlates the intergroup differences were considered not to be of toxicological significance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No significant macroscopic effects were detected in animals of either sex treated with 10, 25 and 75 mg/kg bw/day.
The following macroscopic findings observed were without treatment-related histopathological correlates and were considered to be incidental and unrelated to treatment. One male treated with 10 mg/kg bw/day had an enlarged heart, one male treated with 25 mg/kg bw/day had increased pelvic space in the right kidney, one female treated with 25 mg/kg bw/day had sloughing in the stomach, one non-recovery male treated at 75 mg/kg bw/day had three hard masses approximately 5 mm by 5 mm in the jejunum and one recovery male previously treated with 75 mg/kg bw/day had purple discoloration of the colon.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following treatment-related microscopic abnormality was detected:
Stomach: foveolar hyperplasia was evident in the glandular stomach of four males treated with 75 mg/kg bw/day. This was not present in treated females or in males treated with 10 or 25 mg/kg bw/day. In males previously treated with 75 mg/kg bw/day, this finding had completely reversed following the twenty-eight day recovery period.
There were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or the evaluation of the uterus or of follicles and corpora lutea in the ovaries.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Evaluation of Thyroxine (T4) in adult males did not identify any obvious effect of treatment or indication of endocrine disruption at 10, 25 or 75 mg/kg bw/day.
Statistical analysis of the data did not reveal any significant intergroup differences.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at the highest dose level

Critical effects observed:

no

Tables are attached as pdf below.

Conclusions:

The oral administration of ETPPAAc to rats by gavage, at dose levels of 10, 25 and 75 mg/kg bw/day, resulted in microscopic stomach changes in males treated with 75 mg/kg bw/day.
The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 75 mg/kg bw/day for females and 25 mg/kg bw/day for males.
The reduced body weight development and microscopic changes evident in the stomach were considered to be the result of an irritant effect of the test item rather than a true systemic effect and as such was considered adaptive and non-adverse. Therefore, a ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 75 mg/kg bw/day for males. The ‘No Observed Effect Level’ (NOEL) for reproductive/developmental toxicity was considered to be 75 mg/kg bw/day.

Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item ETPPAAc on reproduction (including offspring development), to evaluate some endocrine disruptor relevant endpoints, and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 29 July 2016). It also assesses the ability of the animals to recover from any toxicity over 28 days following the withdrawal of treatment.

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and up to ten weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 10, 25 and 75 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (0.5% Tween/1% Carboxy Methylcellulose) over the same period. Two recovery groups, each of five males and five females, were treated with the high dose (75 mg/kg bw/day) or the vehicle alone for 43 consecutive days and then maintained without treatment for a further twenty-eight days.

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study.

Pairing of non-recovery animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights, ano-genital distance and visible nipple count (male offspring only).

Extensive functional observations were performed on five selected non-recovery males from each dose group after the completion of the pairing phase, and for five selected non-recovery parental females from each dose group on Day 12post partum. Five non-recovery males and females from each dose group were selected for hematology and blood chemistry assessments prior to termination. Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13post partum, for thyroid hormone analysis; samples from non-recovery adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all nonrecovery treated females including controls through pre-pairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning on the day of termination for all treated females.

Adult males were sacrificed on Day 44 or 45, followed by the sacrifice of all surviving offspring and surviving adult females on Days 13 and 14post partum, respectively. Any female which did not produce a pregnancy or litter was sacrificed around the same time as littering females. Any female which did not show positive evidence of mating and did not produce a pregnancy was sacrificed around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed. All offspring were examined externally; where external observations were detected an internal necropsy was performed.

Following forty-three days of treatment, recovery group animals were maintained without treatment for a further twenty-eight days. In addition, hematological and blood chemical assessments were performed on all recovery group animals at the end of the treatment-free period. These animals were then subjected to a gross necropsy and histopathological examinations of selected tissues was performed.

 

Results…….

Adult Responses

Mortality

There were no treatment-related deaths.

One female treated with 75 mg/kg bw/day was sacrificedin extremison Day 14 due to severe tilting of the head to the left and the inability to walk or right itself. No findings were apparent at macroscopic examination; at histopathology, minimal cortical hypertrophy was apparent in the adrenal gland thus indicating a minor stress-related change. Although the cause of death was not determined, it was considered not to be attributed to the administration of the test item.

 

Clinical Observations

Incidences of noisy respiration and increased salivation were evident in either sex treated with 75 mg/kg bw/day during the treatment period and to a lesser extent in females treated with 25 mg/kg bw/day. An isolated incidence of noisy respiration was also evident in two females treated with 10 mg/kg bw/day. No such effects were evident in males treated with 25 or 10 mg/kg bw/day.

 

Behavioral Assessment

There were no significant changes in the behavioral parameters at 10, 25 or 75 mg/kg bw/day.

 

Functional Performance Tests

There were no changes in functional performance at 10, 25 or 75 mg/kg bw/day.

 

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores at 10, 25 or 75 mg/kg bw/day.

 

Body Weight

Males treated with 75 mg/kg bw/day showed a slight reduction in body weight gain during the first four weeks of treatment. Recovery was evident thereafter. Body weight gain for females during maturation was comparable to controls; however, a reduction in body weight gain was evident during the first two weeks of gestation. Recovery was evident during the final week of gestation and during lactation. Body weight gain for recovery animals during the treatment-free period was comparable to controls. Body weight gains were unaffected by treatment in both sexes at 25 and 10 mg/kg bw/day.

 

Food Consumption

No effect of treatment at 10, 25 or 75 mg/kg bw/day was seen on food consumption or food conversion efficiency (where applicable) in either sex.

 

Water Consumption

Daily visual assessment of water consumption did not reveal any significant intergroup differences.

 

Reproductive Performance

Estrous Cycle

Assessment of estrous cycles during the pre-pairing phase of the study did not indicate any obvious effect of treatment at 10, 25 or 75 mg/kg bw/day. There were also no significant inter-group differences in the stage of estrus on the day of necropsy.

 

Mating

Mating performance as assessed by the number of paired animals that mated was unaffected by treatment at 10, 25 or 75 mg/kg bw/day.

 

Fertility

There was no obvious effect on fertility, as assessed by the number of females that achieved pregnancy at 10, 25 or 75 mg/kg bw/day.

 

Gestation Lengths

The intergroup distribution of gestation lengths observed during the study did not indicate any obvious effect of treatment at 10, 25 and 75 mg/kg bw/day.

 

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no obvious effect of maternal treatment on implantations, post-implantation loss, live birth, post-natal survival or sex ratio of the offspring from birth to termination (Day 13 of age) at 10, 25 or 75 mg/kg bw/day.

 

Offspring Growth and Development

There was no detrimental effect of treatment with the test item indicated by offspring body weight or body weight gain, ano-genital distance on Day 1post partum, visible nipple count in male offspring on Day 13post partumor clinical signs apparent at 10, 25 or 75 mg/kg bw/day.

 

Laboratory Investigations

Hematology

Assessment of hematology parameters did not indicate any obvious effect of treatment for either sex at 10, 25 or 75 mg/kg bw/day.

 

Blood Chemistry

Assessment of blood chemistry parameters did not indicate any obvious effect of treatment for either sex at 10, 25 or 75 mg/kg bw/day.

 

Pathology

Necropsy

Offspring

No significant macroscopic effects were detected in offspring of either sex treated with 10, 25 and 75 mg/kg bw/day.

 

Adults

No significant macroscopic effects were detected in animals of either sex treated with 10, 25 and 75 mg/kg bw/day.

 

Organ Weights

There were no treatment-related effects detected in the organ weights measured at 10, 25 or 75 mg/kg bw/day.

 

Thyroid Hormone Assessment

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 10, 25 or 75 mg/kg bw/day.

 

Histopathology

The following treatment-related microscopic abnormality was detected:

Stomach:foveolar hyperplasia was evident in the glandular stomach of four males treated with 75 mg/kg bw/day. This was not present in treated females or in males treated with 10 or 25 mg/kg bw/day. In males previously treated with 75 mg/kg bw/day, this finding had completely reversed following the twenty-eight day recovery period.

 

Conclusion

The oral administration of ETPPAAc to rats by gavage, at dose levels of 10, 25 and 75 mg/kg bw/day, resulted in microscopic stomach changes in males treated with 75 mg/kg bw/day.

The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 75 mg/kg bw/day for females and 25 mg/kg bw/day for males.

The reduced body weight development and microscopic changes evident in the stomach were considered to be the result of an irritant effect of the test item rather than a true systemic effect and as such was considered adaptive and non-adverse. Therefore, a ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 75 mg/kg bw/day for males. The ‘No Observed Effect Level’ (NOEL) for reproductive/developmental toxicity was considered to be 75 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethyltriphenylphosphonium acetate

EC Number:

252-743-7

EC Name:

Ethyltriphenylphosphonium acetate

Cas Number:

35835-94-0

Molecular formula:

C20H20P.C2H3O2

IUPAC Name:

ethyltriphenylphosphanium acetate

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han™:RccHan™:WIST

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males: approx. 11 weeks, females: approx. 14 weeks
- Weight at study initiation:
males: 289 to 356g; females: 192 to 246g
- Fasting period before study: no
- Housing: groups of three (non-recovery) or five (recovery) in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
During the pairing phase, non-recovery animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the non-recovery males were returned to their original cages. Mated non-recovery females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) ad libitum
- Water (e.g. ad libitum): mains drinking water ad libitum
- Acclimation period: least two weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 15/h
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% Tween/1% Carboxy Methylcellulose

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared fortnightly and stored at approximately 4 ºC in the dark

- VEHICLE
- Concentration in vehicle: 2, 5, 15 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage:
- Length of cohabitation:
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged (how):
- Any other deviations from standard protocol:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test item formulations were taken on three occasions and analyzed for concentration. The results indicate that the prepared formulations were within 97-103 % of the nominal concentration. Results showed the formulations to be stable for at least twenty-one days when stored refrigerated. Formulations were therefore prepared fortnightly and stored at approximately 4 ºC in the dark.

Analysis was performed by HPLC + UV detection
HPLC: Agilent Technologies 1200 with autosampler
Mobile phase: Methanol + 0.1% Formic acid : 100 mM Ammonium Formate + 0.1% Formic acid (85:15, v/v)
Column: Synergi Polar RP 4µ

Duration of treatment / exposure:

non-recovery animals: 14 d pre-mating, max. 14 d mating phase, throughout pregnancy, 13 d post partum
males: total 44 d
recovery animals: dosed according to dose group continuously up to the point of sacrifice of non-recovery males at which time treatment was discontinued + additional 28 days without treatment

Frequency of treatment:

daily

Details on study schedule:

- F1 parental animals not mated (screening study)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were chosen in collaboration with the Sponsor and were based on the results of previous toxicity work including a Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat (Envigo Study Number: MV50QQ). In this study one female treated with 500 mg/kg bw/day was found dead on Day 1 after dosing and two females from this treatment group were sacrificed in extremis due to excessive clinical signs on Day 1. The remaining animals treated with 500 mg/kg bw/day were sacrificed for animal welfare reasons on Day 1. One male treated with 150 mg/kg bw/day was found dead on Day 11 of treatment and the remaining animals at this level were sacrificed on the same day due to excessive dose level. Reductions in body weight gain and food consumption were evident in either sex at 150 mg/kg bw/day and reductions in body weight gain were evident in either sex treated with 100 mg/kg bw/day. At necropsy, macroscopic stomach findings were evident at 500 mg/kg bw/day and in the male that was found dead at 150 mg/kg bw/day. No effects were evident in animals of either sex treated with 50 mg/kg bw/day.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of treatment and at approximately weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioral toxicity (non-recovery mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum). Functional performance tests were also performed on five selected non-recovery males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
Behavioural assessment: Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 (prior to dosing) for non-recovery animals and then weekly for non-recovery males until termination and weekly for non-recovery females until pairing. During pairing phase non-recovery females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4, 7 and 14 post partum. Recovery animals were weighed on Day 1 (prior to dosing) and then approximately weekly until termination. Body weights were also recorded at terminal sacrifice.

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

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: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily by visual inspection of water bottles for any overt changes

OTHER:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 43 for males and Day 13 post partum for females; recovery group animals after the twenty-eight day treatment-free period (Day 71)
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: No
- How many animals: 5
- Parameters: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices (mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), Total leukocyte count (WBC), Differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT), Reticulocyte count (Retic)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 43 for males and Day 13 post partum for females; recovery group animals after the twenty-eight day treatment-free period (Day 71)
- Animals fasted: No
- How many animals: 5
- Parameters: Urea, Inorganic phosphorus (P), Glucose, Aspartate aminotransferase (ASAT), Total protein (Tot.Prot.), Alanine aminotransferase (ALAT), Albumin, Alkaline phosphatase (AP), Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat), Sodium (Na+), Total cholesterol (Chol), Potassium (K+), Total bilirubin (Bili), Chloride (Cl-), Bile acids, Calcium (Ca++)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: non-recovery mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum; five selected non-recovery males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No.

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily for non-recovery females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle.

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 anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other: T4

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, day 44
- Maternal animals: All surviving animals, 13 d post partum
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Examination of offspring was restricted to a macroscopic external examination except where abnormalities were observed; then an additional internal examination was performed.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
Yes (see table)

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external examinations including the cervical, thoracic, and abdominal viscera.
Examination of offspring was restricted to a macroscopic external examination except where abnormalities were observed; then an additional internal examination was performed.

Thyroid Hormone Analysis
Where possible serum samples were taken from two randomly allocated offspring from each litter on Day 4 post partum (if offspring were of the same sex, samples from the same litter were pooled). If eight or fewer offspring were present in a litter, then no offspring from that litter were sampled on Day 4 post partum.
Where possible, serum samples were taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. Where possible, plasma samples were also taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. If required the number/sex of offspring sampled was altered depending on the litter constituents.

Statistics:

see "Any other information on materials and methods"

Reproductive indices:

Pre-coital Interval
Mating Index
Pregnancy Index
Gestation Length
Parturition Index

Offspring viability indices:

Implantation Losses
Post–implantation loss
Live Birth Index
Viability Index 1 (day 4)
Viability Index 2 (day 13)
Sex Ratio (% males)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The majority of animals of either sex treated with 75 mg/kg bw/day showed increased salivation from Day 10 to Day 42 (males) and from Day 14 to Day 57 (females). Incidences of noisy respiration were also evident in three males and four females from this treatment group between Days 15 and 44 (males) and Day 31 and Day 63 (females). One female treated with 25 mg/kg bw/day showed increased salivation on Days 45 and 50 and another female from this treatment group had noisy respiration on Day 20. At 10 mg/kg bw/day, two females had noisy respiration on Day 20. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and are considered not to represent true systemic toxicity.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no treatment-related deaths.
One female treated with 75 mg/kg bw/day was sacrificed in extremis on Day 14, due to the severity of clinical observations. The animal was noted to have mis-aligned posture with the head tilted to the left from Day 11 and exophthalmos in the left eye from Day 13. By Day 14 the head tilt had become more severe with the head being held in an almost upside down position, loss of righting reflex and increased respiratory rate were also observed. Macroscopic necropsy revealed no abnormalities, although minimal cortical hypertrophy in the adrenal gland was observed at histopathology, indicating a minor stress-related change. The cause of the clinical observations could not conclusively be determined; however, findings of this nature are occasionally seen in laboratory maintained rats and are often attributed to inflammation or infection of the middle or inner ear, which can affect balance. The death was not considered to be attributed to the administration of the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males treated with 75 mg/kg bw/day showed a slight reduction in body weight gain during the first four weeks of treatment, although, statistical significance was not achieved. Improvement was evident thereafter, with body weight gains during Week 6 showing statistically significantly higher values when compared to controls. Body weight gain for recovery animals during the treatment-free period was comparable to controls.
No adverse effect in body weight development was evident in treated females during maturation. Females treated with 75 mg/kg bw/day showed a reduction in body weight gain during the first two weeks of gestation, with cumulative body weight gain between Days 0-14 of gestation being statistically significantly lower than controls. Recovery was evident during the final week of gestation and no adverse effects were seen during lactation.
Body weights and body weight gains were unaffected by treatment in both sexes at 25 and 10 mg/kg bw/day throughout treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No effect on food consumption was evident in treated males.
No effect on food consumption (maturation only) was evident in treated females during maturation, gestation or lactation.

Food efficiency:

no effects observed

Description (incidence and severity):

No effect on food conversion efficiency was evident in treated males.
No effect on food conversion efficiency (maturation only) was evident in treated females during maturation, gestation or lactation.

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

no effects observed

Description (incidence and severity):

Daily visual assessment of water consumption did not reveal any significant intergroup differences.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of hematology parameters did not indicate any obvious effect of treatment for either sex at 10, 25 or 75 mg/kg bw/day.
Mean corpuscular hemoglobin concentration for non-recovery males treated at 75 mg/kg bw/day and all non-recovery treated females were statistically significantly lower than controls (p<0.05 - p<0.01). Reticulocytes for all non-recovery treated males and non-recovery females treated with 75 and 25 mg/kg bw/day were statistically significantly higher (p<0.05 - p<0.01) than controls. All individual values were within historical control ranges and in the absence of any histopathological correlates or in the case of male reticulocyte count, no true dose related response, the intergroup differences were considered not to be of toxicological significance.
Eosinophils and platelet values for males previously treated with 75 mg/kg bw/day were statistically significantly increased (p<0.05) and reduced (p<0.05), respectively, at the end of the treatment free period. The majority of individual values for these parameters were within historical control ranges and in the absence of a similar effect in non-recovery animals at the end of the treatment period, the intergroup differences were considered not to be of toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of blood chemistry parameters did not indicate any obvious effect of treatment for either sex at 10, 25 or 75 mg/kg bw/day.
Non-recovery males treated with 75 mg/kg bw/day showed statistically significant reductions in albumin/globulin ratio (p<0.05), chloride concentration (p<0.05), bilirubin (p<0.01) and alkaline phosphatase (p<0.01). Inorganic phosphorus was statistically significantly increased (p<0.01) for these males and bilirubin continued to be significantly reduced (p<0.05) in recovery males that were previously treated with 75 mg/kg bw/day, following the treatment free period. The majority of individual values for these parameters were within historical control ranges and in the absence of any histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Females previously treated with 75 mg/kg bw/day showed a statistically significantly reduction (p<0.05) in potassium concentration at the end of the treatment free period. All of the individual values were within the historical control range and in the absence of a similar effect in non-recovery females at the end of the treatment period, the intergroup difference was considered not to be of toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse changes in the behavioral parameters at 10, 25 or 75 mg/kg bw/day.
Two females treated with 75 mg/kg bw/day showed noisy respiration during assessments on Day 4 and/or Day 12 of lactation. These observations correlated to the daily clinical signs evident at this dose level and were due to the oral administration of an unpalatable or slightly irritant test item formulation rather than evidence of true systemic toxicity.
Mis-aligned posture as evident in the daily clinical observations was noted during the second week of assessments for the female treated with 75 mg/kg bw/day that was sacrificed in extremis. One control male had pilo-erection during the final week of assessment. In the absence of treatment this was considered incidental.

There were no changes in functional performance at 10, 25 or 75 mg/kg bw/day.

There were no inter-group differences in sensory reactivity scores at 10, 25 or 75 mg/kg bw/day.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following treatment-related microscopic abnormality was detected:
Stomach: foveolar hyperplasia was evident in the glandular stomach of four males treated with 75 mg/kg bw/day. This was not present in treated females or in males treated with 10 or 25 mg/kg bw/day. In males previously treated with 75 mg/kg bw/day, this finding had completely reversed following the twenty-eight day recovery period.
There were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or the evaluation of the uterus or of follicles and corpora lutea in the ovaries.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Evaluation of Thyroxine (T4) in adult males did not identify any obvious effect of treatment or indication of endocrine disruption at 10, 25 or 75 mg/kg bw/day.
Statistical analysis of the data did not reveal any significant intergroup differences.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Assessment of estrous cycles during the pre-pairing phase of the study did not indicate any obvious effect of treatment at 10, 25 or 75 mg/kg bw/day, with all females showing regular cycling. There were also no significant inter-group differences in the stage of estrus on the day of necropsy. With the exception of one female treated with 25 or 10 mg/kg bw/day which showed pro-estrus or met-estrus respectively, all other animals exhibited di-estrus on the day of necropsy.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance as assessed by the number of paired animals that mated was unaffected by treatment at 10, 25 or 75 mg/kg bw/day.
One pair treated with 10 mg/kg bw/day failed to mate, however, this was considered to be incidental and unrelated to treatment.

There was no obvious effect on fertility, as assessed by the number of females that achieved pregnancy at 10, 25 or 75 mg/kg bw/day.
One control female and one female treated with 25 mg/kg bw/day showed evidence of pregnancy (implantation sites in the uterus) but no litter was observed to be born. One female treated with 10 mg/kg bw/day was non-pregnant. No changes were observed at histopathology in the females or paired males to account for the lack of pregnancy, therefore, these were considered incidental and unrelated to treatment.

The intergroup distribution of gestation lengths observed during the study did not indicate any obvious effect of treatment at 10, 25 and 75 mg/kg bw/day.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no adverse effects at the highest dose level

2

Dose descriptor:

NOEL

Remarks:

reproduction

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no effects at the highest dose level

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

Effect levels (P1)

Remarks on result:

not measured/tested

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at treatment of 10, 25 or 75 mg/kg bw/day.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no obvious effect of maternal treatment on implantations, post-implantation loss, live birth or post-natal survival of the offspring from birth to termination (Day 13 of age) at 10, 25 or 75 mg/kg bw/day. Sex ratio for the offspring was similar to control in all treated groups and did not indicate any selective effect of maternal treatment on survival for either sex at any of the dosages investigated.
Statistical analysis of the data did not reveal any significant intergroup differences.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no detrimental effect of treatment with the test item indicated by offspring body weight or body weight gain at 10, 25 or 75 mg/kg bw/day.
Statistical analysis of the data did not reveal any significant intergroup differences.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

There was no detrimental effect of treatment with the test item indicated by ano-genital distance on Day 1 post partum or visible nipple count in male offspring on Day 13 post partum at 10, 25 or 75 mg/kg bw/day.
Statistical analysis of the data did not reveal any significant intergroup differences.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related macroscopic abnormalities were detected in interim death or terminal sacrifice offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Other effects:

no effects observed

Description (incidence and severity):

Evaluation of Thyroxine (T4) in offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 10, 25 or 75 mg/kg bw/day.
Statistical analysis of the data did not reveal any significant intergroup differences.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

There was no obvious effect of maternal treatment on implantations, post-implantation loss, live birth or post-natal survival of the offspring from birth to termination (Day 13 of age) at 10, 25 or 75 mg/kg bw/day. Sex ratio for the offspring was similar to control in all treated groups and did not indicate any selective effect of maternal treatment on survival for either sex at any of the dosages investigated.
Statistical analysis of the data did not reveal any significant intergroup differences.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no effects at the highest dose level

Results: F2 generation

Effect levels (F2)

Remarks on result:

not measured/tested

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

TABULAR SUMMARY REPORT OF EFFECTS ON REPRODUCTION/DEVELOPMENT

Observations		Dose Level (mg/kg bw/day)
		0 (control)	10	25	75
Paired animals	n	12M 12F	12M 12F	12M 12F	11M 11F
Females showing evidence of copulation	n	12	11	12	11
Pregnant females	n	12@	10	12@	11
Conception Days 1-4	n	11	10	10	10
Conception Day 13	n	0	0	1	1
Gestation = 22 Days	n	2	2	3	1
Gestation = 22½ Days	n	4	3	4	7
Gestation = 23 Days	n	4	5	2	3
Gestation = 23½ Days	n	1	0	2	0
Dams with live young born	n	11	10	11	11
Dams with live young at Day 13 post partum	n	11	10	11	11
Implants/dam	mean	12.6	13.1	13.6	13.7
Live offspring/dam at Day 1 post partum	mean	11.4	12.1	11.9	12.5
Live offspring/dam at Day 4 BC post partum	mean	11.3	12.1	11.6	12.3
Live offspring/dam at Day 4 AC post partum	mean	9.5	10.3	9.8	10.5
Live offspring/dam at Day 7 post partum	mean	9.5	10.3	9.8	10.5
Live offspring/dam at Day 13 post partum	mean	9.5	10.3	9.8	10.5
Sex ratio: % males at Day 1 post partum	mean	47.3	56.2	50.3	51.3
Sex ratio: % males at Day 4 BC post partum	mean	46.8	56.2	49	50.9
Sex ratio: % males at Day 4 AC post partum	mean	53.3	60	57.6	53.3
Sex ratio: % males at Day 7 post partum	mean	53.3	60	57.6	53.3
Sex ratio: % males at Day 13 post partum	mean	53.5	60	57.6	53.3
Litter weight (g) at Day 1 post partum	mean	66.5	71.4	67.74	70.55
Litter weight (g) at Day 4 BC post partum	mean	97.05	103.96	98.11	99.97
Litter weight (g) at Day 4 AC post partum	mean	82.52	88.47	83	86.03
Litter weight (g) at Day 7 post partum	mean	129.63	137.17	132.32	135.51
Litter weight (g) at Day 13 post partum	mean	250.04	263.69	251.71	258.95
Male offspring weight (g) at Day 1 post partum	mean	6.05	6.13	5.87	5.86
Male offspring weight (g) at Day 4 BC post partum	mean	8.88	8.94	8.7	8.49
Male offspring weight (g) at Day 4 AC post partum	mean	8.88	8.93	8.7	8.47
Male offspring weight (g) at Day 7 post partum	mean	13.88	13.84	13.79	13.28
Male offspring weight (g) at Day 13 post partum	mean	26.81	26.49	26.17	25.38
Female offspring weight (g) at Day 1 post partum	mean	5.75	5.71	5.56	5.54
Female offspring weight (g) at Day 4 BC post partum	mean	8.61	8.41	8.29	8.15
Female offspring weight (g) at Day 4 AC post partum	mean	8.59	8.34	8.32	8.16
Female offspring weight (g) at Day 7 post partum	mean	13.5	12.98	13.37	12.79
Female offspring weight (g) at Day 13 post partum	mean	26.33	25.08	25.66	24.46
LOSS OF OFFSPRING/DAM
Pre-natal (implantations minus live births)
0	n	4	6	0	6
1	n	3	0	7	2
2	n	2	3	2	1
3	n	1	0	1	1
4	n	1	1	0	0
5	n	0	0	1	0
6	n	0	0	0	1
Post natal (live births minus offspring alive on Day 13 post partum) – Excluding offspring culled on Day 4 post partum
0	n	9	10	8	8
1	n	2	0	3	3

Further results are attached as pdf below.

Applicant's summary and conclusion

Conclusions:

The oral administration of ETPPAAc to rats by gavage, at dose levels of 10, 25 and 75 mg/kg bw/day, resulted in microscopic stomach changes in males treated with 75 mg/kg bw/day.
The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 75 mg/kg bw/day for females and 25 mg/kg bw/day for males.
The reduced body weight development and microscopic changes evident in the stomach were considered to be the result of an irritant effect of the test item rather than a true systemic effect and as such was considered adaptive and non-adverse. Therefore, a ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 75 mg/kg bw/day for males. The ‘No Observed Effect Level’ (NOEL) for reproductive/developmental toxicity was considered to be 75 mg/kg bw/day.

Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item ETPPAAc on reproduction (including offspring development), to evaluate some endocrine disruptor relevant endpoints, and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 29 July 2016). It also assesses the ability of the animals to recover from any toxicity over 28 days following the withdrawal of treatment.

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and up to ten weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 10, 25 and 75 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (0.5% Tween/1% Carboxy Methylcellulose) over the same period. Two recovery groups, each of five males and five females, were treated with the high dose (75 mg/kg bw/day) or the vehicle alone for 43 consecutive days and then maintained without treatment for a further twenty-eight days.

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study.

Pairing of non-recovery animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights, ano-genital distance and visible nipple count (male offspring only).

Extensive functional observations were performed on five selected non-recovery males from each dose group after the completion of the pairing phase, and for five selected non-recovery parental females from each dose group on Day 12post partum. Five non-recovery males and females from each dose group were selected for hematology and blood chemistry assessments prior to termination. Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13post partum, for thyroid hormone analysis; samples from non-recovery adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all nonrecovery treated females including controls through pre-pairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning on the day of termination for all treated females.

Adult males were sacrificed on Day 44 or 45, followed by the sacrifice of all surviving offspring and surviving adult females on Days 13 and 14post partum, respectively. Any female which did not produce a pregnancy or litter was sacrificed around the same time as littering females. Any female which did not show positive evidence of mating and did not produce a pregnancy was sacrificed around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed. All offspring were examined externally; where external observations were detected an internal necropsy was performed.

Following forty-three days of treatment, recovery group animals were maintained without treatment for a further twenty-eight days. In addition, hematological and blood chemical assessments were performed on all recovery group animals at the end of the treatment-free period. These animals were then subjected to a gross necropsy and histopathological examinations of selected tissues was performed.

 

Results…….

Adult Responses

Mortality

There were no treatment-related deaths.

One female treated with 75 mg/kg bw/day was sacrificedin extremison Day 14 due to severe tilting of the head to the left and the inability to walk or right itself. No findings were apparent at macroscopic examination; at histopathology, minimal cortical hypertrophy was apparent in the adrenal gland thus indicating a minor stress-related change. Although the cause of death was not determined, it was considered not to be attributed to the administration of the test item.

 

Clinical Observations

Incidences of noisy respiration and increased salivation were evident in either sex treated with 75 mg/kg bw/day during the treatment period and to a lesser extent in females treated with 25 mg/kg bw/day. An isolated incidence of noisy respiration was also evident in two females treated with 10 mg/kg bw/day. No such effects were evident in males treated with 25 or 10 mg/kg bw/day.

 

Behavioral Assessment

There were no significant changes in the behavioral parameters at 10, 25 or 75 mg/kg bw/day.

 

Functional Performance Tests

There were no changes in functional performance at 10, 25 or 75 mg/kg bw/day.

 

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores at 10, 25 or 75 mg/kg bw/day.

 

Body Weight

Males treated with 75 mg/kg bw/day showed a slight reduction in body weight gain during the first four weeks of treatment. Recovery was evident thereafter. Body weight gain for females during maturation was comparable to controls; however, a reduction in body weight gain was evident during the first two weeks of gestation. Recovery was evident during the final week of gestation and during lactation. Body weight gain for recovery animals during the treatment-free period was comparable to controls. Body weight gains were unaffected by treatment in both sexes at 25 and 10 mg/kg bw/day.

 

Food Consumption

No effect of treatment at 10, 25 or 75 mg/kg bw/day was seen on food consumption or food conversion efficiency (where applicable) in either sex.

 

Water Consumption

Daily visual assessment of water consumption did not reveal any significant intergroup differences.

 

Reproductive Performance

Estrous Cycle

Assessment of estrous cycles during the pre-pairing phase of the study did not indicate any obvious effect of treatment at 10, 25 or 75 mg/kg bw/day. There were also no significant inter-group differences in the stage of estrus on the day of necropsy.

 

Mating

Mating performance as assessed by the number of paired animals that mated was unaffected by treatment at 10, 25 or 75 mg/kg bw/day.

 

Fertility

There was no obvious effect on fertility, as assessed by the number of females that achieved pregnancy at 10, 25 or 75 mg/kg bw/day.

 

Gestation Lengths

The intergroup distribution of gestation lengths observed during the study did not indicate any obvious effect of treatment at 10, 25 and 75 mg/kg bw/day.

 

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no obvious effect of maternal treatment on implantations, post-implantation loss, live birth, post-natal survival or sex ratio of the offspring from birth to termination (Day 13 of age) at 10, 25 or 75 mg/kg bw/day.

 

Offspring Growth and Development

There was no detrimental effect of treatment with the test item indicated by offspring body weight or body weight gain, ano-genital distance on Day 1post partum, visible nipple count in male offspring on Day 13post partumor clinical signs apparent at 10, 25 or 75 mg/kg bw/day.

 

Laboratory Investigations

Hematology

Assessment of hematology parameters did not indicate any obvious effect of treatment for either sex at 10, 25 or 75 mg/kg bw/day.

 

Blood Chemistry

Assessment of blood chemistry parameters did not indicate any obvious effect of treatment for either sex at 10, 25 or 75 mg/kg bw/day.

 

Pathology

Necropsy

Offspring

No significant macroscopic effects were detected in offspring of either sex treated with 10, 25 and 75 mg/kg bw/day.

 

Adults

No significant macroscopic effects were detected in animals of either sex treated with 10, 25 and 75 mg/kg bw/day.

 

Organ Weights

There were no treatment-related effects detected in the organ weights measured at 10, 25 or 75 mg/kg bw/day.

 

Thyroid Hormone Assessment

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 10, 25 or 75 mg/kg bw/day.

 

Histopathology

The following treatment-related microscopic abnormality was detected:

Stomach:foveolar hyperplasia was evident in the glandular stomach of four males treated with 75 mg/kg bw/day. This was not present in treated females or in males treated with 10 or 25 mg/kg bw/day. In males previously treated with 75 mg/kg bw/day, this finding had completely reversed following the twenty-eight day recovery period.

 

Conclusion

The oral administration of ETPPAAc to rats by gavage, at dose levels of 10, 25 and 75 mg/kg bw/day, resulted in microscopic stomach changes in males treated with 75 mg/kg bw/day.

The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 75 mg/kg bw/day for females and 25 mg/kg bw/day for males.

The reduced body weight development and microscopic changes evident in the stomach were considered to be the result of an irritant effect of the test item rather than a true systemic effect and as such was considered adaptive and non-adverse. Therefore, a ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 75 mg/kg bw/day for males. The ‘No Observed Effect Level’ (NOEL) for reproductive/developmental toxicity was considered to be 75 mg/kg bw/day.