Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol

Endpoint summary

Description of key information

- combined repeat dose toxicity study with the reproduction/developmental screening test (OECD 422): NOAEL >= 1000 mg/kg bw/day (m/f)

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Feb - 13 Oct 2022

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:

adopted in 2016

Deviations:

yes

Remarks:

Minor deviations in pup identification, access to water, dosing, clinical observations, food consumption, body weight, and organ weights. No impact to the overall integrity of the study or the interpretation of the study results and conclusions.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 11 - 12 weeks for males; 13 - 14 weeks for females
- Weight at study initiation: 271 - 317 g for males; 190 - 240 g weeks for females
- Fasting period before study: no
- Housing: Up to 5 animals of the same dosing group together in polycarbonate cages (Makrolon, MIV type, height 18 cm)
- Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany; Pellets ad libitum, except during designated procedures.
- Water: Municipal tap water; Freely available to each animal via water bottles. One female did not have access to water for a maximum of 24 h.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): >= 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 21 Feb - 21 Jun 2022

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were divided into aliquots and where dispensed on each dosing occasion. The test item was formulated (w/w) and homogenized to visually acceptable levels. It was formulated at least weekly and filled out in daily portions. The dose formulation was stored at 4 °C.

VEHICLE
- Justification for use and choice of vehicle: Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20326206) demonstrated that the test item was stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
- Amount of vehicle: The dose volume for each animal was based on the most recent body weight measurement. The dose formulations were stirred continuously during dose administration and doses were given using a plastic feeding tube.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using a laboratory validated analytical procedure.

The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 90 - 110% of target concentration). No test material was detected in the Group 1 formulation.
The formulations of Group 2 and Group 4 were homogeneous (i.e., coefficient of variation ≤ 10%). Homogeneity of Group 3 was not analyzed.

Duration of treatment / exposure:

Males: 7 days a week for a minimum of 28 days, including at least 2 weeks of treatment prior to mating and during the mating period (up to and including the day before scheduled necropsy).

Females: 7 days a week for at least 14 days prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females were not be dosed during littering.

Pups: Not to be treated directly but could potentially be exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.

Frequency of treatment:

once daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of the Dose Range Finder (Test Facility Reference No. 20326207).
- Fasting period before blood sampling for clinical biochemistry: All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 h before necropsy. Females were not be fasted before necropsy.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before the first administration of the test item and weekly during the treatment period (observed for mortality at least twice daily beginning upon arrival through termination/release; except on days of receipt and necropsy where frequency was at least once daily).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily, up to the day prior to necropsy. No clinical observations were recorded on dosing Day 2 for two animals and dosing Day 3 for one animal.

BODY WEIGHT: Yes
- Time schedule for examinations: On Day 1 of treatment (prior to dosing) and weekly
thereafter. Mated females: On Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
The terminal body weight of one female was not recorded.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption for one female was not determined during post-coitum Day 0 - 4. One male was dosed on the day of necropsy.

WATER CONSUMPTION AND COMPOUND INTAKE: No

HEMATOLOGY AND COAGULATION:
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Only for males
- How many animals: 5/sex/group
- Parameters checked: white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cells, red blood cells, reticulocytes, red blood cell distribution, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Only for males
- How many animals: 5/sex/group
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate

THYROID HORMONE:
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Only for males
- How many animals: 5/sex/group
- Parameters checked: thyroxine (T4), thyroid-stimulating hormone (TSH)

OPHTHALMOSCOPIC EXAMINATION: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during the treatment period.
- Dose groups that were examined: all doses
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore and hind limb grip strength, locomotor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY: Yes
The following tissues were weighed: brain, epididymis, adrenal gland, parathyroid gland, prostate gland, seminal vesicle, thyroid gland, heart, kidney, liver, ovaries, spleen, testes, thymus, uterus/cervix
Organ weights of one male and brain weight of one male were not recorded.

The following tissues were prepared for microscopic evaluation: bone marrow (sternum), femur, sternum, brain, epididymis, eye, adrenal gland, mammary gland, pituitary gland, prostate gland, seminal vesicle, thyroid gland, gut associated lymphoid tissue, heart, kidney, cecum, colon, rectum, liver, lung, mandibular lymph node, mesenteric lymph node, skeletal muscle, sciatic nerve, ovaries, duodenum, ileum, jejunum, spinal cord, spleen, stomach, testes, thymus, trachea, urinary bladder, uterus/cervix, vagina

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test material.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight of treated animals was similar to the control level over the treatment period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No test material-related changes were noted in hematological parameters in males up to 300 mg/kg bw/day and females up to 1000 mg/kg bw/day. In males at 1000 mg/kg bw/day, lower neutrophil count was noted. No corroborating pathological findings were observed, and therefore this change was considered non-adverse.
Remaining differences in hematology parameters, regardless of statistical significance, were considered not test material-related based on the absence of a dose response or general overlap of individual values with the range of control values.
Coagulation parameters of treated rats were considered not to have been affected by treatment with the test material.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No test material-related changes were noted in clinical chemistry parameters in males up to 300 mg/kg bw/day.
The following changes in clinical biochemistry parameters distinguished treated animals from
control animals: Higher urea levels in males at 1000 mg/kg bw/day; Higher total bilirubin (not statistically significant) for females at 100, 300 and 1000 mg/kg bw/day. No corroborating pathological findings were observed, and therefore these changes were considered non-adverse.
Any other significant changes were considered unrelated to the treatment in the absence of a dose-related response or because individual values were within range of control values.

Thyroid hormone
Serum levels of T4 in males were considered unaffected by treatment with the test material up to 1000 mg/kg bw/day.

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study:
- thyroid hormones
- accessory sex organ weight and histopathology
- anogenital distance
- sex organ weight and histopathology
- oestrous cyclicity
- genital abnormalities
- nipple development
- thyroid weight and histopathology
- vaginal smears
- adrenal weight and histopathology
- brain weight
- fertility
- fetal development
- gestation length
- litter size
- litter viability
- litter / pup weight
- number of implantations, corpora lutea
- number of live births
- pituitary histopathology
- post-implantation survival
- reproduction
- sex ratio

For details, please refer to the RSS in IUCLID section 7.8.1.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were considered not to have been affected by treatment with the test material.
Lower hind leg grip strength was noted in females at 100, 300, and 1000 mg/kg bw/day (not always statistically significant). In the absence of a dose-relationship and as mean values are within the historical control range, this was considered not related to treatment with the test material.
Motor activity was considered not to be affected by treatment with the test material. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
In females at all dose levels mean total movements and ambulations were lower (not statistically significant), which could be attributed to one or two animals per group. In the absence of clear dose relationship and as the control means were slightly high, this was considered unrelated to the treatment with the test material.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test material-related alterations in organ weights.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test material-related gross observations.
All the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related microscopic observations.
All the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test material-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to and including the highest dose tested

Critical effects observed:

no

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:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, of Regulation (EC) No. 1907/2006.

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

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

The repeat dose and reproductive/developmental toxicity of esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol was investigated in a study performed according to OECD guideline 422 under GLP conditions (Charles River, 2022e). The registration substance was administered once daily, 7 days/week, via gavage to groups of 10 Wistar rats per sex at doses of 100, 300, and 1000 mg/kg bw in corn oil. A control group received the vehicle alone. Males were treated for at least 28 days, including at least 2 weeks of treatment prior to mating and during the mating period (up to and including the day before scheduled necropsy). Females were treated for at least 14 days prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. In-life observations and measurements included mortality, clinical observations, body weights, food consumption, water consumption, and functional tests. Clinical pathology included haematology, coagulation, clinical chemistry, and thyroid hormone measurements. Terminal procedures included necropsy, tissue collection, organ weights, histology processing and microscopic evaluation of certain tissues. Parameters and results in relation to reproduction/developmental toxicity are provided in the endpoint summary in IUCLID section 7.8.

No mortality occurred during the study period. No treatment-related effects were seen in clinical signs, body weight, food/water consumption, and functional tests.

No treatment-related effects were seen in coagulation parameters, thyroid hormone levels, macroscopic findings, organ weights, and microscopic observations. A lower neutrophil count was observed in males at 1000 mg/kg bw/day. Higher urea levels were observed in males at 1000 mg/kg bw/day. Higher total bilirubin for females was observed at 100, 300, and 1000 mg/kg bw/day. As no corroborative findings were observed in any other measure, the above changes were considered non-adverse.

Based on the available data, no changes that could be considered adverse were observed at any of the dose levels investigated. Therefore, the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was concluded to be 1000 mg/kg bw/day, for both male and female parental animals (for information on reproduction/developmental toxicity please refer to IUCLID section 7.8).

Justification for classification or non-classification

According to Regulation (EC) No. 1272/2008 the data on repeat dose toxicity are conclusive but not sufficient for classification.