Triglycerides, C16-C18 (even numbered) and C18 (unsaturated), maleated

Endpoint summary

Description of key information

Repeated dose toxicity:
- Glycerol: NOAEL = 10000 mg/kg bw/day (NTP 2 year feeding study)
- Fatty acids, C14-18 and C16-18-unsatd., maleated.: NOAEL = 1000 mg/kg bw/day (OECD 422)

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:

combined repeated dose and reproduction / developmental screening

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

According to the guideline and under GLP tested with the source substance CAS 85711-46-2. As the tested substance is a breakdown product of the target substance, this study is considered valid for read-across. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

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: Wistar Crl:WI rats
- Source:Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
- Age at study initiation: 11 weeks
- Weight at study initiation: males: Males: 350 g – 382 g, Females: 183 g - 218 g
- Fasting period before study: overnight prior to treatment
- Housing: 5 animals of the same sex and group/cage with the exception of the mating and gestation/delivery period, when they
were paired or individually housed, respectively. (cage: Type II and/or III polycarbonate)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20,1-25
- Humidity (%): 36-70
- Air changes (per hr):15-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 mL/kg bw

The test material and the vehicle was warmed up on a water bath to 50 C for approximately 10 minutes separately and mixed up on a hot stirrer plate. Once a suitable formulation was obtained, the container was removed from the plate. Pending administration to the animals, the dose formulations were stirred on a magnetic stirrer at room temperature and were protected from light.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of WS400104 formulations for concentration and homogeneity was performed using validated HPLC method (CiToxLAB study code 11/352-316AN). The concentration analysis was performed on 3 occasions, during the first, fourth and last weeks of the treatment period. Recovery of WS400104 from propylene glycol ranged between 92% and 106% (92% - 104%, 97% - 106%, 96% - 102% respectively to the three analysis performances).

Duration of treatment / exposure:

Main males: 35 days (14 days pre-mating, 14 days mating/post-mating period followed by an additional week)
Main females: ca. 47 days (14 days pre-mating, for up to 7 days mating period, through gestation til PPD 4)
Satellite females (nulliparous and nonpregnant): 35 days

Frequency of treatment:

daily, 7 days/week

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg b.w./day
Basis:
actual ingested

No. of animals per sex per dose:

Main groups: 12 animals/sex/dose
Satellite group (20 femal rats): 5 animals/dose

Offspring were not dosed.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose finding toxicity study with WS400104 (administered via oral gavage to Wistar rats for 7 consecutive days at dose levels of 100, 300 and 1000 mg/kg bw) showed no effects (7.5.1 Dose Range Finding Study 7 days_WS400104).

Based on these results, the dose levels selected for the main study were 0, 100, 300 and 1000 mg/kg bw/day.

This study (OECD Guideline 422) was conducted to examine both repeated dose toxicity and reproductive/developmental toxicity as an OECD screening combined study . Therefore, animals initially entering the study were divided into toxicity subgroup animals (Satellite females) and reproductive subgroup animals (Main females and males), whereby 5 of the 12 Main males (used for pairing) per dose group formed the toxicity male Subgroup A.

Positive control:

No

Observations and examinations performed and frequency:

see Table 1a-c: Schedule and Dosing scheme ("Any other information on material and methods incl. tables")
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least weekly
Observations in a standard arena: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule: All adult Main and Satellite animals were weighed on Day 0, afterwards at least weekly and at termination.
Parent females were weighed on gestation Days GD 0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition), and PPD5 (before termination).

FOOD CONSUMPTION: yes
-Time schedule: at least weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy: Day 35
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (overnight)
- How many animals: Subgroup A (5 main males/group), satellite females (5/group)
- Parameters examined: bloodcells, coagulation (APIT, PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy: Day 35
- Animals fasted: Yes (overnight)
- How many animals:S ubgroup A (5 main males/group), satellite females (5/group)
- Parameters examined: Glucose, T-Bilirubin, Urea, Cholesterol, Creatinine, Phosphorus, Sodium, Potassium, Calcium, Chlorid, Total protein, Albumin, AST, ALT, ALKP, GGT, Bile acids.

URINALYSIS: Yes
- Time schedule for collection of urine: prior to necropsy
- Metabolism cages used for collection of urine: Yes (ca. 16 h)
- How many animals: Subgroup A (5 main males/group), satellite females (5/group)
- Parameters examined: Leukocyte, Nitrit, pH, protein, Glucose, Urobilinogen, Bilirubin, Ketones, Erythrocytes, Specific gravity, Sediment, Volume, Colour/Appearance.

NEUROBEHAVIOURAL EXAMINATION, Neurotoxicity: yes, FOB
- Time schedule: during the last exposure week (on Day 32)
- Dose groups that were examined: Main animals, 5 males/group, “subgroup A” and Toxicology Satellite females of all dose levels
- Battery of functions tested: sensory activity / grip strength / motor activity /general physical condition and behaviour of animals

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Animals: each adult animal, pups (PND4)
- Time schedule for the adults: on Day 35, i.e. one day after the last treatment following overnight period food deprivation, or PND5

HISTOPATHOLOGY: Yes, see Table 1-3 ("Any other information on materials and methods incl. tables")

Statistics:

Performance with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test,the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Effects on prothrombin time in mid and high dose groups not considered toxicologically significant.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Effects observed were regarded as physicological variation.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Observations in high dose groups (1000 mg/kg b.w./day). In the stomachs of several male and female rats hyper/parakeratosis of the non-glandular gastric mucosa was observed at minimal to mild degree.

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no clinical signs related to treatment.

BODY WEIGHT AND WEIGHT GAIN
There was no adverse effect of test material on body weight or body weight gain. Body weights were comparable to the control in all treated groups.

FOOD CONSUMPTION
There was no adverse effect of treatment on food consumption. The individual values remained within the normal ranges.

HAEMATOLOGY
There were no test material related adverse effects at any dose level, all parameters falling with the normal physiological ranges for the age, strain and sex of rats on test.
Slightly lower prothrombin time (PT) values were measured in males at 300 and 1000 mg/kg (Mid and High dose), compared to the control. The mean values were lower by approximately 9% and differences were statistically significant. As the mean and individual values remained within the normal
ranges, the differences were of low magnitude, therefore the finding was not considered toxicologically significant or to reflect an adverse effect of WS400104.

CLINICAL CHEMISTRY
There were no toxicologically significant effects at any dose levels.

URINALYSIS
There was no effect of treatment noted during urinalysis.

NEUROBEHAVIOUR
There were no treatment related effects. There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups. There was no effect of treatment noted during the assessment of foot splay or grip strength.

NECROPSY
There were no macroscopic findings related to treatment.
Observations of some changes were considered incidental.

ORGAN WEIGHTS
There were no toxicologically significant effects on organ weights.
Slightly lower weights of kidneys were recorded for males at 300 and 1000 mg/kg bw/day (Mid and High dose). The changes were not associated with any findings in clinical pathology or microscopic changes and were regarded as physiological variation.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Test material related microscopic findings were found at 1000 mg/kg bw/day (High dose) in stomach, in the form of hyper/paraceratosis of the nonglandular gastric mucosa. This minimal to mild multifocal change occurred in 4 of 5 males and 2 of 5 females.
There were no microscopic findings related to treatment at 100 or 300 mg/kg bw/day.

Dose descriptor:

NOAEL

Remarks:

for systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

In the report the NOAEL of WS400104 administered by oral route to Wistar rats for 35 consecutive days is considered to be the mid-dose level of 300 mg/kg bw/day.
However, based on the fact that no systemic effects were observed in the study and considering that the effects seen in the pars non-glandularis of the the stomach are indicative of local irritation and that analogous structures do not exist in man it is concluded that the NOAEL of this subacute oral toxicity study is 1000 mg/kg/d for systemic toxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

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 repeated dose toxicity is assessed by using the results of a combined repeated dose and reproduction / developmental screening study performed according to OECD 422 with the source substance Fatty acids, C14-18 and C16-18-unsatd., maleated. Groups of twelve males and females received daily dosages of 100, 300 or 1000 mg/kg test substance bw/day by gavage, respectively. Males were treated for 35 days (14 days pre-mating, 14 days mating/post-mating period followed by an additional week), whereas females received the test material for approx. 47 days (14 days pre-mating, for up to 7 days mating period, through gestation until PPD 4), respectively. Examinations of the parental animals included detailed clinical observations, body weight, food consumption, haematology, clinical chemistry, urinalysis and neurobehavioural examination. At the end of study gross pathology and histopathology was performed. The only substance-related effect have been noted in animals treated with the dose levels of 1000 mg/kg bw/day, where a minimal/mild hyper/paraceratosis of the nonglandular gastric mucosa in stomach in 4 of 5 males and 2 of 5 females have been found. However, this finding was considered to be indicative of local irritation in a structure of the stomach that does not exist in man. Consequently the NOAEL was found to be 1000 mg/kg bw/day, the highest dose tested due to the absence of any systemic effects up to the highest dose.

In a dietary study with natural and synthetic glycerol, goups of 22 rats (Long-Evans)/sex/treatment received 5, 10 and 20% glycerol (natural or synthetic) in their diet (males 2000, 4000 and 8000 mg/kg bw; females 2500, 5000 and 10000 mg/kg bw) for 2 years. Routine clinical observations were made, and bodyweight and food consumption was determined weekly. Deviations from the current OECD guideline included the absence of clinical chemistry investigation and a limited range of haematological and urinary analyses were performed. A limited range of organs was investigated at necropsy and the liver, spleen, adrenals, small intestine, gonads and urinary bladder were examined microscopically. Glycogen and fat content of the liver was determined in surviving rats from the 0 and 20 % dose groups. No individual data were reported. For high dosed animals treatment was discontinued after 1 year (reason not stated in report, presumably as an ‘interim’ assessment for carcinogenicity. No data on mortality and clinical observations were reported. Food consumption was slightly increased in males treated with 5 and 10% natural glycerol. Incidental observations considered by the report-authors to be without relationship to treatment included: bronchiectasis, pneumonia, pulmonary abcesses, hydronephrosis and pyelonephritis. Based on the absence of overt adverse effects and despite the results were not described in detail, the NOAEL could be determined to be 10,000 mg/kg bw/day.

Because the tested substances Fatty acids, C14-18 and C16-18-unsatd., maleated and glycerol represent the breakdown products after hydrolysis by pancreatic lipases, the same result have to be expected for High oleic sunflower oil, maleated.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP-Guideline study with source substance Fatty acids, C14-18 and C16-18-unsatd., maleated.

Justification for classification or non-classification

The available data on repeated dose toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.