Fatty acids, vegetable-oil, Me esters, sulfurized

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

FROM 23 MAR 2018 TO 12. FEB 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, vegetable-oil, Me esters, sulfurized

EC Number:

276-337-4

EC Name:

Fatty acids, vegetable-oil, Me esters, sulfurized

Cas Number:

72102-30-8

IUPAC Name:

Fatty Acids, vegetable-oil, Me-Esters, sulfurized

Test material form:

liquid

Details on test material:

- Density: 0.95 g/mL
- Batch no.: 71012767

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat is the species and strain of choice because it is accepted by many regulatory authorities and there is ample experience and background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 to 8 weeks
- Weight at study initiation: 220-230 g for males and 183-191 g for females
- Fasting period before study: none
- Housing: The animals will be housed in a limited access rodent facility. Animal room controls will be set to maintain temperature and relative humidity at 22°C ± 2°C and 55% ± 15% respectively; actual conditions will be monitored, recorded and the records retained. There will be approximately 15 to 20 air changes per hour and the rooms will be lit by artificial light for 12 hours each day.
From arrival to mating, animals will be housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5x38x20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material will be provided inside suitable bedding bags and changed at least twice a week.
During mating, animals will be housed one male to one female in clear polysulfone cages measuring 42.5x26.6x18.5 cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray will hold absorbent material which will be inspected and changed daily.
After mating, the males will be re-caged as they were before mating. The females will be transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5x26.6x18.5 cm). Nesting material will be provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) will be provided as necessary. Nesting material will be changed at least 2 times a week.
- Diet: ad libitum throughout the study. Blood collection was performed for hormone determination from all animals at termination under condition of food deprivation. Blood samples for haematology, clinical chemistry and coagulation were collected by random selection from 5 males and 5 females (females with viable litters) under condition of food deprivation.
- Water: ad libitum
- Acclimation period: 27 days

DETAILS OF FOOD AND WATER QUALITY:
A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milanese (MI), Italy)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 April 2018 To: 04 July 2018

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered orally, by gavage. The oral route was selected as it is a possible route of exposure of the test item in man.
The test item was administered orally by gavage at a dose volume of 10mL/kg body weight.
Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): daily
- Mixing appropriate amounts with (Type of food): not applicable
- Storage temperature of food: not stated

VEHICLE
- Justification for use and choice of vehicle: The test item is based on fatty acids and therefore almost insoluble in water. Due to that fact, vegetable oils would interfere during the analytical determination. CMC 0.5% was chosen to guarantee a stable suspension in water.
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle: The test item will be administered orally by gavage at a dose volume of 10 mL/kg body weight. The dose will be administered to each animal on the basis of the most recently recorded body weight and the volume administered will be recorded for each animal.
- Lot/batch no.: SLBR5921V
- Purity: not stated

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in RTC Study no. A3060 in the range from 10 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in RTC SOPs for suspensions (r > 0.98; accuracy 85-115%; precision CV < 10%).
In RTC Study no. A3060, a 26 hour stability at room temperature and a 10 day stability at+5°C ± 3°C were verified in the range from 10 to 100 mg/mL. According to RTC SOPs, suspensions are considered to be stable if concentration and homogeneity, after the defined period of storage, are still acceptable (85%-115% for concentration and CV < 10% for homogeneity).
The proposed formulation procedure for the test item was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) in RTC Study no. A3060 to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in RTC SOPs for concentration (85-115%) and homogeneity (CV < 10%).

For further details please refer to the report "A3060 - Validation of the analytical method and formulation procedure in CMC" in the attached background material.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily, 7 days each week for 2 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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:

The toxic effects on Sprague Dawley rats of both sexes after repeated dosing with Fatty Acids, vegetable-oil, Me-Esters, sulfurized, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation of the offspring were investigated. The vehicle was carboxymethyl cellulose 0.5% (CMC 0.5%). All doses were administered at a constant volume of 10mL/kg body weight.

The experimental design was as follows:
Group Treatment Number of animals
(mg/kg bw/day)
1 0 10M+10F
2 100 10M+10F
3 300 10M+10F
4 1000 10M+10F

Males were treated for 14 days prior to pairing and during pairing with females until the day before necropsy, for a total of 29/30 days. Females were treated for 14 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for a total of 51-64 days.

Dose levels were selected in consultation with the Sponsor based on information from a preliminary, non-GLP compliant study.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.
- Cage side observations checked in Table 2; Appendix 2 of the attached study report.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination.
The tests included observation of Removal (from cage), Handling reactivity, Lachrymation, Palpebral closure, Salivation, Piloerection, Rearing, Spasms, Myoclonia, Mobility impairment, Arousal (animal activity), Vocalisation, Stereotypies, Unusual respiratory pattern, Bizarre behaviour, Urination, Defecation, Tremors and Gait

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20 post coitum.
Dams were also weighed on Days 1, 4, 7 and 13 post partum and just prior to necropsy.

FOOD CONSUMPTION:
The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from Day 1 of dosing. Individual food consumption for the females was measured on gestation Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum.

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: Not specified

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Not specified
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: 5M + 5F
- Parameters checked in Tables 9 and 10 of the attached study report were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Not specified
- Animals fasted: Yes
- How many animals: 5M + 5F
- Parameters checked: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride, Inorganic phosphorus

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during the study, towards the end of treatment
- Dose groups that were examined: all
- Battery of functions tested: sensory activity, grip strength, motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (for details see table 25 of the attached study report no. X0910)
The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.

All females were examined also for the number of visible implantation sites (pregnant animals) and the number of corpora lutea (pregnant animals).

Pups
All pups found dead in the cage were examined for external and internal abnormalities.
All culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection. All live pups sacrificed at Day 14 post partum were killed and examined for external abnormalities and sex confirmation by gonads inspection. All pups with abnormalities were retained in a 10% neutral buffered formalin.

Organ weights
Parental animals
From all animals the organs indicated in section 4.5.7were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.
Pups at Day 14 post partum
Thyroid was weighed from one male and one female from each litter (where possible the same pup selected for serum hormone determination) and preserved for possible histopathological examination. The thyroid weight was determined after fixation.

HISTOPATHOLOGY: Yes (for details see table 26 of the attached study report no. X0910)
Tissues fixed and preserved
Samples of Abnormalities; Adrenal glands; Bone marrow (from sternum); Brain (cerebrum, cerebellum, medulla/pons); Caecum; Clitoral gland; Colon; Duodenum; Epididymides; Eyes; Femur with joint; Heart; Ileum; Jejunum (including Peyer’s patches); Kidneys; Liver; ;Lungs (including mainstem bronchi); Lymph nodes – cervical; Lymph nodes – mesenteric; Mammary area – Females; Mammary area –Males; Nasal cavity (not microscopically examined since no signs of toxicity or target organ were involvement); Oesophagus; Ovaries with oviducts; Parathyroid glands (weighed and preserved with thyroid gland); Pituitary gland; Penis; Prostate gland (dorsolateral and ventral); Rectum; Sciatic nerve; Seminal vesicles with coagulating glands; Skeletal muscle; Spinal column (not microscopically examined since no signs of toxicity or target organ were involvement); Spinal cord (cervical, thoracic, lumbar); Spleen; Stomach; Testes; Thymus (where present); Thyroid; Trachea; Urinary bladder; Uterus – cervix; Vagina were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves, testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol).

Other examinations:

not performed

Statistics:

Standard deviations were calculated as appropriate. For variables such as body weight, food consumption, clinical pathology parameters and organ weight the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings were carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of theWilliams test. The criterion for statistical significance was p<0.05.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs occurred during the study.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No changes of toxicological relevance in body weight and body weight gain were observed during the study in the treated animals, when compared to controls. The statistically significant decrease (of approximately 5%) recorded in high dose group of males on Day 1 of treatment was considered incidental, as well as the statistically significant decrease detected in body weight gain at the same group of males before pairing.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Animal no. X0910062 (1000 mg/kg/day) showed mild leucocytosis. Compared with mean controls, the increase was 2.1 fold. Due to the minimal incidence, this finding cannot be conclusively attributed to treatment. The statistically significant differences of mean corpuscular haemoglobin concentration recorded between control and treated females were of minimal severity (up to 2%), therefore they were considered to be of no toxicological importance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant differences recorded between control and treated animals (globulin and albumin/globulin ratio in males, cholesterol and glucose in females) were not dose-related, therefore they were considered to be incidental.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.
No treatment-related alterations in motor activity, grip strength, landing footsplay and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No changes were observed on terminal body weight of treated animals, when compared to the controls. No relevant changes were seen in the absolute and relative organ weights of treatment groups of both sexes, when compared to control data. The decreased absolute and relative kidneys weight of mid- and/or high dose male groups was not considered of toxicological significance, since the histopathological evaluation of this organ was comparable to control animals.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Animals that completed the treatment period did not show macroscopic changes that could be considered treatment-related. The sporadic changes such as pelvic dilatation of kidneys observed in two high dose males (nos. X0910062 and X0910064) and one low dose female (no. X0910021) and small thymus in females from each dosed group [nos. X0910033 (Group 2), X0910043 (Group 3) and X0910071 (Group 4)] were considered spontaneous and incidental under our experimental conditions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not specified

Other effects:

not examined

Effect levels

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:

behaviour (functional findings)

body weight and weight gain

clinical signs

gross pathology

haematology

mortality

organ weights and organ / body weight ratios

Remarks on result:

other: No signs of treatment-related toxicity were observed following treatment with Fatty Acids, vegetable-oil, Me-Esters, sulfurized, when administered to rats by oral route at dose levels of 100, 300 and 1000 mg/kg/day, at any of the dose levels investigated.

Remarks:

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general, reproductive and developmental toxicity was considered to be 1000 mg/kg/day for males and females.

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, no signs of treatment-related toxicity were observed following treatment with Fatty Acids, vegetable-oil, Me-Esters, sulfurized, when administered to rats by oral route at dose levels of 100, 300 and 1000 mg/kg/day, at any of the dose levels investigated. Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general, reproductive and developmental toxicity was considered to be 1000 mg/kg/day for males and females.

Executive summary:

In conclusion, no signs of treatment-related toxicity were observed following treatment with Fatty Acids, vegetable-oil, Me-Esters, sulfurized, when administered to rats by oral route at dose levels of 100, 300 and 1000 mg/kg/day, at any of the dose levels investigated. Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general, reproductive and developmental toxicity was considered to be 1000 mg/kg/day for males and females.