Ethyltriphenylphosphonium acetate

Endpoint summary

Description of key information

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.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

dose-range finding study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

22 August 2017 - 17 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

designed as dose range finding study

GLP compliance:

no

Remarks:

The study was conducted in a facility which operates in accordance with Good Laboratory Practice principles, however no claim of GLP compliance was intended nor is made for this dose range finding study.

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 12 weeks
- Weight at study initiation: 286 to 390 g (males), 205 to 236 g (females)
- Housing: in groups of three by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- 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 supplied from polycarbonate bottles attached to the cage , ad libitum
- Acclimation period: at least five days

DETAILS OF FOOD AND WATER QUALITY:

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

Route of administration:

oral: gavage

Vehicle:

other: 0.5% Tween/1% Carboxy Methylcellulose or Distilled Water

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in 0.5% Tween/1% Carboxy Methylcellulose and for selected dose levels, as a suspension in Distilled Water.

The test item was administered within two hours of it being formulated. It is assumed that the formulation was stable for this duration.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 d

Frequency of treatment:

daily

Dose / conc.:

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

Remarks:

formulation prepared using 0.5% Tween 80/1% Carboxy Methylcellulose

Dose / conc.:

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

Remarks:

formulation prepared using 0.5% Tween 80/1% Carboxy Methylcellulose

Dose / conc.:

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

Remarks:

formulation prepared using 0.5% Tween 80/1% Carboxy Methylcellulose

Dose / conc.:

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

Remarks:

formulation prepared using 0.5% Tween 80/1% Carboxy Methylcellulose

Dose / conc.:

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

Remarks:

formulations prepared using Distilled Water as the vehicle

Dose / conc.:

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

Remarks:

formulations prepared using Distilled Water as the vehicle

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule:
All animals were examined for overt signs of toxicity, ill health or behavioral change immediately before dosing, up to thirty minutes after dosing and one hour after dosing. Additional observations were also made four hours following dosing (not at weekends). All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Days 1, 4, 8, 11 and 15.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was recorded for each cage group for Days 1 to 4, 4 to 8, 8 to 11 and 11 to 15. Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was measured and recorded daily for each cage group.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: cardiac puncture at termination
- Animals fasted: No
- How many animals: all animals that survived to termination
- Parameters: Sodium (Na+), Calcium (Ca++), Potassium (K+), Inorganic Phosphorus (P), Chloride (Cl-)


URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On completion of the dosing period, all surviving animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination and subjected to an internal and external macroscopic examination. Animals that died during the study were also necropsied. The liver, kidneys, stomach and intestines were preserved from all animals in buffered 10% formalin.

HISTOPATHOLOGY: No

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The females treated with 500 mg/kg bw/day (with 0.5% Tween/1% Carboxy Methylcellulose vehicle) that were sacrificed in extremis on Day 1 showed splayed gait, laboured respiration, decreased respiratory rate, prostration, clonic convulsions and ataxia. The remaining female from this treatment group had pilo-erection, hunched posture and ataxia whilst the males from this treatment group had decreased respiratory rate, hunched posture, ataxia and one male also had clonic convulsions and prostration. The animals treated with 500 mg/kg bw/day (with Distilled Water vehicle), that were terminated due to excessive dose level showed pilo-erection, hunched posture and ataxia.
At 150 mg/kg bw/day, animals of either sex showed episodes of increased salivation (Days 1-2 with Distilled Water vehicle and Days 4-10 (males) and Days 4-8 (females) with 0.5% Tween/1% Carboxy Methylcellulose vehicle).
At 100 mg/kg bw/day, animals of either sex showed instances of increased salivation from Day 7 (males) and Day 8 (females) to Day 14.
At 50 mg/kg bw/day, all females and one male showed increased salivation on Day 12 or Day 13 (respectively) only.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two females treated with 500 mg/kg bw/day (with 0.5% Tween/1% Carboxy Methylcellulose vehicle) were sacrificed in extremis due to adverse clinical signs on Day 1. One female treated with 500 mg/kg bw/day (with Distilled Water vehicle) was found dead following dosing on Day 1. The remaining animals from both of these treatment groups were terminated on Day 1 due to excessive dose level. All of these animals had gaseous distension in the stomach and a pale glandular region of the stomach at necropsy.
One male treated with 150 mg/kg bw/day (with 0.5% Tween/1% Carboxy Methylcellulose vehicle) was found dead on Day 11. At necropsy, this male also had gaseous distension in the stomach and a pale glandular region of the stomach. The remaining animals from both of the 150 mg/kg bw/day treatment groups were terminated on this day (treatment Day 8 for the Distilled Water group) due to excessive dose level. These animals did not show any macroscopic abnormalities at necropsy.
There were no further unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 150 mg/kg bw/day (using both vehicles), body weight gains between Days 1 and 4 were comparable to controls. Between Days 4 and 8, body weight gains for these males were lower than controls and two males from the 0.5% Tween/1% Carboxy Methylcellulose vehicle group showed actual body weight losses.
Females treated with 150 mg/kg bw/day (with 0.5% Tween/1% Carboxy Methylcellulose vehicle) showed body weight gains that were comparable to controls between Days 1 and 8; however, between Days 8 and 11, body weight gains were reduced. Females treated with 150 mg/kg bw/day (with Distilled Water vehicle) showed actual body weight losses between Days 1 and 4. Between Days 4 and 8, body weight for one female from this treatment group remained static and the other female showed a body weight loss.
At 100 mg/kg bw/day, males showed slightly lower body weight gains between Days 1 and 4. Improvement was evident between Days 4 and 11, however, actual body weight losses were evident in these males between Days 11 and 15. Overall body weight gain for males was 42% lower than controls. One female from this treatment group showed an actual body weight loss between Days 1 and 4, whilst the other female remained static in body weight. Between Days 4 and 15, both females either remained static in weight or only gained a small amount of weight. Overall body weight gain for females was 69% lower than controls.
No adverse effects were evident in body weight gains in animals of either sex treated with 50 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Males treated with 150 mg/kg bw/day (with 0.5% Tween/1% Carboxy Methylcellulose vehicle) and females treated with 150 mg/kg bw/day (with Distilled Water vehicle) showed lower food consumptions. No such effects were detected in the remaining animals treated with 150 mg/kg bw/day from either group.

No overall adverse effect on food consumption was noted in animals of either sex treated with 100 or 50 mg/kg bw/day.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Fluctuations in food conversion efficiency were evident in animals of either sex treated with 150 mg/kg bw/day (both vehicles) and generally followed the reductions in body weight gain seen in these animals.

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

effects observed, non-treatment-related

Description (incidence and severity):

Males treated with 150 mg/kg bw/day (with Distilled Water vehicle) showed an increase in water consumption. No such effects were detected in the remaining animals treated with 150 mg/kg bw/day from either group.
Males treated with 50 mg/kg bw/day showed an increase in overall water consumption, however, no adverse effect on water consumption was detected in animals of either sex treated with 100 mg/kg bw/day or in females treated with 50 mg/kg bw/day.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Blood samples were taken from all animals (excluding both 500 mg/kg bw/day groups).
There were no obvious treatment-related effects detected in the blood chemical parameters measured.
Chloride concentration for treated animals of either sex and sodium concentration for treated females were comparable to control values.
Potassium concentration for treated males was lower than controls; however, true dose related responses were not evident. Potassium concentration for treated females did not show a true effect of treatment. Both of the 150 mg/kg bw/day dose groups showed lower values compared to controls, whilst the 100 mg/kg bw/day dose group showed higher values and the 50 mg/kg bw/day dose group was comparable to controls.
Calcium concentration for treated animals of either sex and sodium concentration for 150 mg/kg bw/day males showed slight variations to control values; however, true dose related responses were not evident.
Phosphorus levels for treated animals of either sex showed variations to control values; however, true dose related responses were not evident.

Urinalysis findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic abnormalities were detected in scheduled kill animals of either sex treated with 100 or 50 mg/kg bw/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Dose descriptor:

dose level: high dose for full OECD TG 422 study

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Dose descriptor:

dose level: mid dose for full OECD TG 422 study

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Dose descriptor:

dose level: low dose for full OECD TG 422 study

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Critical effects observed:

not specified

Conclusions:

The oral (gavage) administration of ETPPAAc at dose levels of 50, 100, 150 and 500 mg/kg bw/day for a period of up to fourteen consecutive days, resulted in treatment-related effects in animals of both sexes treated with 500, 150 and 100 mg/kg bw/day. The necessary early termination of both 500 and 150 mg/kg bw/day dose groups and reduction in body weight gain at 100 mg/kg bw/day were sufficient to exclude these dose levels from further investigation. Therefore, dose levels of 0 (Control), 10, 25 and 75 mg/kg bw/day are recommended for use in the planned Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test.

Executive summary:

ETPPAAc was administered by gavage to seven groups, of either two or three male and two or three female Wistar Han™:RccHan™:WIST strain rats, for up to fourteen consecutive days, at initial dose levels of 50 mg/kg bw/day (with 0.5% Tween/1% Carboxy Methylcellulose vehicle), 150 mg/kg bw/day (with 0.5% Tween/1% Carboxy Methylcellulose vehicle), 500 mg/kg bw/day (with 0.5% Tween/1% Carboxy Methylcellulose vehicle) and 500 mg/kg bw/day (with Distilled Water vehicle). Another dose group (150 mg/kg bw/day with Distilled Water vehicle) was added to the study following the early termination of both of the 500 mg/kg bw/day dose groups and a further dose group (100 mg/kg bw/day (with 0.5% Tween/1% Carboxy Methylcellulose vehicle) was added to the study following the early termination of both of the 150 mg/kg bw/day dose groups. A control group of three males and three females was dosed with vehicle alone (0.5% Tween/1% Carboxy Methylcellulose).

Clinical signs, body weight change, dietary intake and waterconsumption were monitored during the study. At termination, blood samples were taken for selected blood chemical analysis and all animals were subjected to a gross necropsy examination (including retention of selected tissues) at termination.

Mortality

Two females treated with 500 mg/kg bw/day (with 0.5% Tween/1% Carboxy Methylcellulose vehicle) were sacrificedin extremisdue to adverse clinical signs on Day 1. One female treated with 500 mg/kg bw/day (with Distilled Water vehicle) was found dead following dosing on Day 1. The remaining animals from both of these treatment groups were terminated on Day 1 due to excessive dose level. All of these animals had gaseous distension of the stomach and a pale glandular region of the stomach at necropsy.

One male treated with 150 mg/kg bw/day (with 0.5% Tween/1% Carboxy Methylcellulose vehicle) was found dead on Day 11. At necropsy, this male also had gaseous distension of the stomach and a pale glandular region of the stomach. The remaining animals from both of the 150 mg/kg bw/day treatment groups were terminated on this day (treatment Day 8 for the Distilled Water group) due to excessive dose level. These animals did not show any macroscopic abnormalities at necropsy.

There were no further unscheduled deaths.

Clinical Observations

The females treated with 500 mg/kg bw/day (with 0.5% Tween/1% Carboxy Methylcellulose vehicle) that were sacrificedin extremison Day 1 showed splayed gait, respiratory pattern changes, prostration, clonic convulsions and ataxia. The remaining female from this treatment group had pilo-erection, hunched posture and ataxia whilst the males from this treatment group had decreased respiratory rate, hunched posture, ataxia and one male also had clonic convulsions and prostration. The animals treated with 500 mg/kg bw/day (with Distilled Water vehicle) showed pilo-erection, hunched posture and ataxia.

At 150 mg/kg bw/day (using both vehicles), animals of either sex showed episodes of increased salivation.

At 100 and 50 mg/kg bw/day, animals of either sex showed instances of increased salivation.

Body Weight

At 150 mg/kg bw/day (using both vehicles), body weight gains between Days 1 and 4 were comparable to controls. Between Days 4 and 8, body weight gains for these males were lower than controls. Females treated with 150 mg/kg bw/day (with 0.5% Tween/1% Carboxy Methylcellulose vehicle) showed body weight gains that were comparable to controls between Days 1 and 8; however, between Days 8 and 11, body weight gains were reduced. Females treated with 150 mg/kg bw/day (with Distilled Water vehicle) showed actual body weight losses between Days 1 and 4 and reduced gains between Days 4 and 8. 

At 100 mg/kg bw/day, males showed slightly lower body weight gains between Days 1 and 4; showed improvement between Days 4 and 11 and then showed actual body weight losses between Days 11 and 15. One female from this treatment group showed an actual body weight loss between Days 1 and 4, whilst the other female remained static. Between Days 4 and 15, both females either remained static in weight or only gained a small amount of weight. Overall body weight gains in either sex were lower than controls.

No adverse effects were evident in body weight gains in animals of either sex treated with 50 mg/kg bw/day.

Food Consumption

Males treated with 150 mg/kg bw/day (with 0.5% Tween/1% Carboxy Methylcellulose vehicle) and females treated with 150 mg/kg bw/day (with Distilled Water vehicle) showed lower food consumptions. No such effects were detected in the remaining animals treated with 150 mg/kg bw/day from either group.

No overall adverse effect on food consumption was noted in animals of either sex treated with 100 or 50 mg/kg bw/day.

 

Water Consumption

Males treated with 150 mg/kg bw/day (with Distilled Water vehicle) showed an increase in water consumption. No such effects were detected in the remaining animals treated with 150 mg/kg bw/day from either group. 

Males treated with 50 mg/kg bw/day showed an increase in overall water consumption. No adverse effect on water consumption was detected in animals of either sex treated with 100 mg/kg bw/day or in females treated with 50 mg/kg bw/day.

Blood Chemistry

Blood samples were taken from all animals (excluding both 500 mg/kg bw/day groups). There were no obvious treatment-related effects detected in the blood chemical parameters measured.

Necropsy

No macroscopic abnormalities were detected in scheduled kill animals of either sex treated with 100 or 50 mg/kg bw/day.

Conclusion

The oral (gavage) administration of ETPPAAc at dose levels of 50, 100, 150 and 500 mg/kg bw/day for a period of up to fourteen consecutive days, resulted in treatment-related effects in animals of both sexes treated with 500, 150 and 100 mg/kg bw/day. The necessary early termination of both 500 and 150 mg/kg bw/day dose groups and reduction in body weight gain at 100 mg/kg bw/day were sufficient to exclude these dose levels from further investigation. Therefore, dose levels of 0 (Control), 10, 25 and 75 mg/kg bw/day are recommended for use in the planned Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The study was designed to investigate the systemic toxicity and potential adverse effects of 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 twenty-eight 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 forty-three 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 12 post 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 non-recovery 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.

 

Adult Responses

Mortality

There were no treatment-related deaths.

One female treated with 75 mg/kg bw/day was sacrificed in extremis on 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 1 post partum, visible nipple count in male offspring on Day 13 post partum or 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.

 

 

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.

Justification for classification or non-classification

Based on the available data, the substance ETPPAAc does not need to be classified and labelled according to the CLP Regulation (EC) No 1272/2008 with respect to repeated dose toxicity.