Fatty acids, coco, esters with 3,3'-oxybis[1,2-propanediol]

Endpoint summary

Description of key information

A NOAEL of 1000 mg/kg bw/day could be derived from a reliable combined repeated dose and reproduction screening study in rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

examined within the range of a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 September 2016 to 07 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening
Test” adopted on 29 July 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch No.of test material: ESD0021912
- Expiration date of the lot/batch: 17.03.2020
- Purity test date: 100 % [w/w]
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility and stability of the test substance in the vehicle: Vehicle used was corn oil and test item
formulation was stable for 24 hours at room temperature
FORM AS APPLIED IN THE TEST: Liquid

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation: Male: 220 to 260 g
Female: 200 to 240 g
- Housing: standard polypropylene cage (size: L 430 x B 285 x H 150 mm)
- Diet (e.g. ad libitum): Teklad Certified (2014SC) Global 14 % Protein Rodent Maintenance Diet -
Pellet (Manufactured by Envigo)
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed
to ultraviolet rays in Aquaguard water filter with purifier
- Acclimation period: 29 September 2016 to 17 October 2016
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 to 22.9oC
- Humidity (%): 48 to 69%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle
IN-LIFE DATES: From: 18 October 2016 To: 24 December 2016

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Test item formulation was prepared daily before administration

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During first week and week 5 of treatment

Duration of treatment / exposure:

Males: 37 days
Females: Approximately 70 days including premating, mating, gestation and lactation periods.

Frequency of treatment:

Once daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

Main group: 12 males and 12 females per dose
Recovery group: 5 males and 5 females per dose (control and high dose groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses of 0, 100, 300 and 1000 mg/kg body weight for vehicle control/ vehicle control recovery, low dose, mid dose and high dose/ high dose recovery groups respectively were selected in consultation with the sponsor based on the results of dose range finding study of test item when administered through oral gavage for a period of 14 consecutive days to Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 1944).
These doses were selected as the test item Hostastat FE 20 LIQ when administered orally to Sprague Dawley rats once daily for 14 consecutive days did not reveal any treatment related effects at all the tested doses (100, 300 and 1000 mg/kg body weight).

- Rationale for animal assignment: The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ± 20%

Positive control:

no

Observations and examinations performed and frequency:

Parental animals: Observations and examinations
CAGE SIDE OBSERVATIONS: Yes
- Once daily throughout the experimental period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly
- Signs noted included, but not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT: Yes
- Time schedule for examinations: The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The females were weighed on gestation days 0, 7, 14
and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The recovery group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was measured for main group animals once a week during premating and for main group males during post mating period. Feed consumption was not measured during mating period for both males and females. Thereafter, feed consumption for females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13. Feed consumption was measured for recovery group animals once a week throughout the experimental period.

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmological examination was carried out once before treatment for all animals, during end of the dosing period for males (shortly prior to scheduled sacrifice) and during lactation period for females (shortly prior to scheduled sacrifice) of vehicle control and high dose main group animals and during last week for recovery group animals. The examination was not extended to lower dose groups as there were no treatment related changes noted in high dose group animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Dams at termination (lactation day 14), adult males at termination (after completion of 37 days of treatment)
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: five males and five females randomly selected from each main group and from all recovery group animals
- Parameters checked: at least according to guideline

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Dams at termination (lactation day 14), adult males at termination (after completion of 37 days of treatment)
- Animals fasted: Yes
- How many animals: five males and five females randomly selected from each main group and from all recovery group animals
- Parameters checked: at least according to guideline, including T4 levels

URINALYSIS: Yes
- Time schedule for collection of urine: At termination of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Albumin, Leucocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: five males and five females, randomly selected from each group towards the end of the dosing period for males (shortly prior to scheduled sacrifice
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity / grip strength / motor activity / Home Cage Observations / Handling Observations / Open Field Observations / Neuromuscular Observations / Physiological Observation (Rectal temperature)

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Estrous cyclicity (parental animals)
Oestrus cycles were monitored during the acclimatization to evaluate its normal oestrus cyclicity (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment. Vaginal sm
ears were monitored daily from the beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which may induce
pseudopregnancy. Oestrus cyclicity was also monitored on the day of sacrifice for females. Recovery group females were not evaluated for oestrus cyclicity.

Sacrifice and pathology:

Postmortem examinations (parental animals)
SACRIFICE
- Male Animals: All Animals after 37 days of treatment
- Female Animals: All animals were sacrificed on Lactation day 14.
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera: Yes
HISTOPATHOLOGY / ORGAN WEIGHTS
Control and high dose groups examined, examination range at least according to guideline, with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure

Statistics:

One-way ANOVA with Dunnett’s post test
Kruskal-Wallis followed by the Mann-Whitney test if differences were indicated
Chi-square test/ Fischer's Exact Test
The Parameters include Body weight , Change in body weight, Feed consumption, Copulatory interval, Gestation length, Hematology, Clinical chemistryAbsolute organ weights, Relative organ weights, FOB, Body temperature, Defecation, Urination, Rearing, Grip strength, Litter weights , Pup weight , Preimplantation loss, Pre natal loss, Post natal loss, Total No. of early/late resorptions/dam, Corpora lutea/dam, Implantations/dam, No. of pups/dam, Sex ratio, Mean litter size, Mean pup weight, Pregnancy rate, No. of dams with/without live pups, No. of dams with/without dead pups, No. of litters with/without resorptions.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity observed at any of the tested dose main groups of either sex during premating period and mating period, during post mating period in males, during gestation/lactation period in females and throughout the experimental period in recovery group animals.
The detailed clinical examination of animals did not reveal any treatment related changes at any of the main and recovery group animals of either sex.

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity observed at any of the tested dose main groups of either sex during premating period and mating period, during post mating period in males, during gestation/lactation period in females and throughout the experimental period in recovery group animals.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment related changes in mean body weight and percent change in body weight with respect to day 1 at any of the main and recovery group animals of either sex

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatment related changes noted in feed consumption at any of the doses at any of the main and recovery group animals of either sex.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular changes observed in vehicle control and high dose group animals during the ophthalmological examination for males conducted at the end of the dosing period, for females during the lactation period and for recovery group animals at the end of recovery period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in haematology at any of the tested doses of either sex. However, statistically significant decrease in prothrombin time in low, mid and high dose group females and statistically significant increase in activated prothrombin time in the recovery group females when compared with vehicle control was noted. This change is considered as incidental and not treatment related, as the values are within historical control range and same changes not observed in males.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in clinical chemistry at any of the doses tested in both the sex.

However, in main group males statistically significant decrease in alkaline phosphatase level at low dose and statistically significant increase in sodium level at mid and high dose noted when compared with vehicle control. In females, statistically significant increase in cholesterol at mid dose, statistically significant increase in albumin at high dose and statistically significant increase in calcium at high dose were noted when compared with vehicle control.

In recovery males, statistically significant increase in creatinine, total protein, albumin, and chloride and statistically significant decrease in phosphorous at recovery when compared with vehicle control and in recovery females statistically significant increase in creatinine, cholesterol, albumin and sodium and statistically significant decrease in phosphorous at recovery when compared with vehicle control was noted.

These changes are considered as an incidental and not treatment related, as the values are within historical control range and is due to biological variations.

Statistically significant decrease in T4 levels in high dose males when compared with vehicle control group were noted when compared with vehicle control group.
The variation observed in the T4 levels could not be attributed as treatment related as there were no microscopic observations noted in thyroid gland and no effects were noted in thyroid weight at all the tested dose groups. This significant variation could be due to the increased T4 levels in vehicle control group males (Animal No. Rb8893: 99.593 ng/mL and Animal No. Rb8898: 99.416 ng/mL) and low dose group male (Animal No. Rb8905: 110.605 ng/mL). The T4 levels obtained across the groups are within historical control range. Hence, the variation observed in the T4 levels is considered to be incidental

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed at any of the dose tested. However, statistically significant decrease in urine volume at mid dose (males), significant decrease in urobilinogen in low, mid and high dose males and significant increase in pH at the end of recovery (females) was noted. This change is considered as an incidental and not treatment related, as the values are within historical control range.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The detailed clinical examination of animals did not reveal any treatment related changes at any of the main and recovery group animals of either sex.
There were no changes observed in the neurological/functional examination conducted for main group males (prior to scheduled sacrifice), for main group females during the lactation period (prior to scheduled sacrifice) and for all recovery group animals at the end of recovery period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in the absolute and relative organ weights at any of the doses tested of both the sex in main and recovery group animals.
However, statistically significant decrease in absolute brain and heart weight high dose males, statistically significant increase in absolute and relative spleen and heart weight in mid dose females and statistically significant increase in absolute PSC weight (Prostate+Seminal vesicles with coagulating glands) in low dose males were noted when compared with vehicle control. This change can be considered as incidental and not treatment related, as there were no gross and microscopic findings were noted in these organs.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes (both external and internal) observed at any of the doses

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment related histopathological findings noticed in high dose group animals.
Testes, which were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all expected associations.
All other observed histopathological lesions in high dose group animals, considered as spontaneous and incidental to Sprague Dawley rats of this particular age as they lack in consistency.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no changes observed in the oestrus cyclicity of females at any of the doses tested during pre-mating treatment, mating treatment and on lactation day 14.
There were no treatment related changes in gestation and lactation body weight, percent change in gestation and lactation body weights, feed consumption during gestation period and lactation period, uteri observations, litter and pup weights, anogenital distance of pups (see in detail in chapter 7.8.1).

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of treatment-related adverse effects

Key result

Critical effects observed:

no

The dose formulation analysis for concentration verification was performed during week 1 and 5 of treatment period and the results were within acceptable limits.

Conclusions:

Based on the results of this study, the NOAEL was 1000 mg/kg body weight when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), and to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 under the experimental conditions employed in this study.

Executive summary:

The test item, was evaluated for possible adverse effects following repeated oral dosing to males for 37 days and to females, two weeks pre-mating period, during mating, pregnancy

(gestation) and up to lactation day 13 to evaluate effects of test item on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus,

parturition, and early neonatal development.

A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups. G1 (control), G2 (low dose), G3 (mid dose) and G4 (high dose) groups consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle[Corn oil], the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of100, 300 and 1000 mg/kg body weight for low dose, mid dose and high dose/high dose recovery groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight. The main group males were treated for two weeks pre-mating, during mating and up to the day before

sacrifice during post-mating period (total of 37 days of treatment). The main group females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after

which the pups were sacrificed on lactation Day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed). The recovery group males and females were treated until the

first scheduled sacrifice of dams and kept without treatment for a further 16 days observation. The dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results were within acceptable limits.

All animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight and feed consumption. Ophthalmological examination was carried out once before treatment

for all animals, duringend of the dosing period for males (shortly prior to scheduled sacrifice) and during lactation period for females (shortly prior to scheduled sacrifice)for vehicle control and high dose

group animals andduring last weekfor recovery group animals.Neurological/Functional examination was performed for five males and five females, randomly selected from each group towards the end of the

dosing period for males (shortly prior to scheduled sacrifice), during lactation period for females (shortly prior to scheduled sacrifice) and from both the recovery group animals towards the end of the recovery

period (shortly prior to scheduled sacrifice).

The clinical pathology (haematological and clinical chemistry) examinations were conducted in five males and five females from each main group (randomly selected) andfor all recovery group animals.

Urinalysis was performed from the five randomly selected males of each main group during the last week of the treatment period and for all recovery animals at termination.

Gross pathology and organ weighing were performed on day 38 for males, on lactation day 14 for dams and on day 68 for recovery group animals. The number of corpora lutea, implantation sites and resorptions for dams were recorded.

The animals did not reveal any clinical signs of toxicity. No mortality or morbidity was observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight,

feed consumption, ophthalmoscopic examination, neurological/functional examination, haematology, clinical chemistry, urine analysis nor in organ weights (both absolute and relative) were observed in both

the sex of main and recovery group animals. The dams did not reveal any treatment related changes in gestation and lactation body weight, percent change in gestation and lactation body weights, feed consumption during gestation period and lactation period, uteri observations, litter and pup weights, anogenital distance of pups. The animals did not reveal any treatment related gross pathological and histopathological findings.

Based on the results discussed, the No Observed Adverse Effect Level (NOAEL) of the test item was found to be 1000 mg/kg body weight when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), and to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 under the experimental conditions employed in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable study, GLP-compliant and according to OECD guideline 422

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

Mode of Action Analysis / Human Relevance Framework

In the absence of any evidence for species specific effects or modes of action possibly observed effects would be regarded as relevant for humans.

Additional information

In this GLP-compliant study according to OECD guideline 422 the test item was administered daily (gavage) to male and female Sprague-Dawley rats at dose levels of 100, 300 and 1000 mg/kg bw/day. No effects were observed with respect to parental toxicity, effects on reproduction and development of pups.

Justification for classification or non-classification

As no systemic effects were oberved up to the highest dose (1000 mg/kg bw/day), no classification is required with respect to specific organ toxicity after repeated exposure according to the criteria laid down in Regulation (EC) No 1272/2008 (CLP).