Reaction mass of Fatty acids, tallow, Guerbet reaction Products, Ca salts and Fatty acids, tallow, Guerbet reaction Products, Na salts and Fatty acids, tallow, Guerbet reaction Products

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:

Jan-March 2017

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:

2018

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 (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

73138-58-6, 73138-59-7, 73138-60-0

IUPAC Name:

73138-58-6, 73138-59-7, 73138-60-0

Details on test material:

- Name of test material (as cited in study report): LICOWAX R 21 S FL
- Physical state: Slightly yellow flakes
- Analytical purity: 99.6 % (w/w)

Fatty acids, tallow,
Guerbet reaction Products 73138-58-6 21% (w/w)
Fatty acids, tallow,
Guerbet reaction Products, Ca salts 73138-59-7 48% (w/w)
Fatty acids, tallow,
Guerbet reaction Products, Na salts 73138-60-0 31% (w/w)

- Lot/batch No.: DEF2084336
- Expiration date of the lot/batch: 2018-11-13
- Storage condition of test material: Room temperature, protected from light in the tightly closed original container
- Solubility : ≤ 0.01 g/L (20 °C)

Specific details on test material used for the study:

Batch No DEF2084336
Purity: 99.6%

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Breeding, RCC Laboratories India Private Limited, Hyderabad - 500 078, India
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:14-15 Weeks
- Weight at study initiation: Male : 283.7 to 376.3 g and Female:231.8 to 275.4 g
- Housing: Initially (acclimatization and randomization period), all animals were be housed in groups of two to three per polycarbonate cages (approximate internal dimensions of 365 mm x 202 mm x 180 mm height) with paddy husk bedding. Results of analyses for contaminants will be archived at RCC Laboratories India Private Limited. After randomization, males and females were housed individually. During the mating phase, animals were housed on one male: one female basis within each dose group. After successful mating, the females were returned to cages and housed individually during gestation and lactation.
- Diet (e.g. ad libitum): Teklad Certified Global 14% Protein Rodent Maintenance Diet (Lot No. 2014C-061716MA) from ENVIGO was provided ad libitum.
- Water (e.g. ad libitum): Aquaguard filtered tap water was provided ad libitum.
- Acclimation period: 12. Jan. - 2. Feb. 2017

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 to 22.9°C
- Humidity (%): 53 to 63 %
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 12. Jan. To: 8. April 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Refined ground nut oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on preliminary solubility testing
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose Concentration: Samples for each dose formulation (low, intermediate and high) were taken before start of the treatment and at termination of study.
Stability: Samples for each dose formulation (low, intermediate and high) were taken before start of the treatment (0 hour) and after 6 hours of treatment.
Homogeneity: Samples in triplicate for each dose formulation (low, intermediate and high) were taken from lower, middle and top layers for homogeneity test. Samples in triplicate for each dose formulation were taken for dose concentration before start of treatment and at termination of study.
One control sample for each dose formulation was taken and treated in a similar way.

Duration of treatment / exposure:

Main groups:
Males: 42 days
Females: premating, mating, gestation periods and up to day 13 post partum

Satellite groups:
continuously up to day 50 without mating and dosing was stopped on first schedule sacrifice of dam

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

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:

Main groups: 12
Satellite groups: 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Base on results of dose range finding study
- Rationale for selecting satellite groups: to determine persistence of potential effects

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATION: Yes
Males: before dosing, once weekly (except during mating)
Females: before dosing and once weekly (except during mating), gestation days 7, 14, 20 and days 4, 13 post partum
recovery period: once weekly

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: weekly
Females:
Premating - weekly
Gestation - days 0, 7, 14 and 20
Lactation - days 1, 4 and 13
Recovery - weekly

FOOD CONSUMPTION:
Males: once weekly until termination
Females:
Premating - once weekly
Gestation - days 7, 14 and 20
Lactation - days 0, 4 and 13
Recovery:weekly

HAEMATOLOGY: Yes (Five males and five females were selected randomly from each test and control group prior to termination (Day before the terminal sacrifice). All the animals of satellite group were screened at the end of recovery period)
Blood samples for hematology and clinical biochemistry analysis were collected from animals selected for repeated dose toxicity screen and animals of satellite group. The animals were placed in metabolic cages and fasted overnight before blood sampling and urine collection, but allowed access to water ad libitum.
Blood sampling:
Males - Day 43
Females - Day 14 post partum
Satellite group: at the end of recovery period (Day 65)
Analysed parameters:
erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglaobin concentration, platelet (thrombocyte) count, total leukocyte count, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes (Five males and five females were selected randomly from each test and control group prior to termination (Day before the terminal sacrifice). All the animals of satellite group were screened at the end of recovery period)
Blood samples for hematology and clinical biochemistry analysis were collected from animals selected for repeated dose toxicity screen and animals of satellite group. The animals were placed in metabolic cages and fasted overnight before blood sampling and urine collection, but allowed access to water ad libitum.
Blood sampling:
Males - Day 43
Females - Day 14 post partum
Satellite group: at the end of recovery period (Day 65)
Analysed parameters:
glucose, urea, creatinine, cholesterol, cholesterol total, triglycerides, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, bilirubin, sodium, potassium, chloride, protein total, albumin, globulin, A/G ratio

Hormone analysis:
-T4 and TSH from
- Two pups per litter on day 4 after birth
- All dams on day 13
- Two pup per sex per litter at termination on day 13
- all adult males at termination


URINALYSIS: Yes
At study termination
Analysed parameters:
Volume, specific gravity, colour, clarity, pH, Erythrocytes, Leukocytes, urobillinogen, Bilirubin, ketone bodies, proteins and glucose

NEUROBEHAVIOURAL EXAMINATION: Yes
Sensory reactivity, Grip Strength and Motor Activity prior to termination


OTHER:
- Repdroductive Indices:
The following data was evaluated
1. Mating performance and fertility (Pre-coital interval, mating index and pregnancy index)
2. Gestation and parturition Data (Gestation length and parturition index)
3. Litter Responses (Pre implantation loss, live/birth index and sex ratio)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All the animals were subjected to gross examination. The male animals were sacrificed by CO2 asphyxiation on day 43 and female animals were sacrificed on day 14 post partum.
Dead pups were subjected to gross examination, whereas live pups were sacrificed at day 13 post partum and gross examination was performed. All animals were weighed and sent to the necropsy.

Organ weights:
The following organ weights were recorded on the scheduled dates of necropsy for five males and five females animals selected for repeated dose toxicity screening and all the animals of satellite group:
Liver, kidney, adrenals, thymus, spleen, brain, heart, testes, epididymides, ovaries

HISTOPATHOLOGY: Yes
Samples of the following tissues and organs were collected from main groups (Randomly selected five males and five females for each sex/ group), and allocation satellite animals at necropsy:
Adrenal glands, Aorta, Bone marrow (Sternum), Brain (3 levels), Cecum, Colon, Duodenum, Epididymides, Heart, Ileum, with Peyer's patches, Jejunum, Kidneys, Liver, Lungs (inflated with NBF at necropsy), Lymph nodes (mesenteric, axillary), Oesophagus, Ovaries, Pancreas, Pituitary, Seminal Vesicle + Prostate, Rectum, Sciatic Nerve, Skeletal muscle, Spinal cord (cervical, mid thoracic, lumbar), Spleen, Stomach, Testes, Thymus, Thyroid, Trachea, Urinary bladder (inflated with NBF at necropsy), Uterus, Gross lesions

Slides of all organs and tissues (listed under necropsy) from all animals of the control (0 mg/kg) and high dose (1000 mg/kg) were processed, embedded in paraffin, cut at approximate thickness of 3 to 5 micrometers, stained with Hematoxylin & Eosin (H & E) and examined microscopically by the Study Pathologist.
The examination revealed treatment related changes in liver of high dose group (1000 mg/kg) which extended the histological preparation of liver in all animals of low (100 mg/kg), intermediate (300 mg/kg) as well as in control satellite (0 mg/kg) and high dose satellite (1000 mg/kg) groups

Statistics:

The statistical methods were used to analyze the body weight, feed consumption and organ weights.
Data are summarized in tabular form. Statistical analysis was performed using statplus program. Statistical analysis of AGD was performed based on individual pup data. Where appropriate, the litter was the unit of analysis. Statistical analysis of pup body weight was based on individual pup data, taking litter size into account.
• The continuous data was checked for Normality with Shapiro-Wilk test and subjected to perform Analysis of Variance (ANOVA) to compare the difference between treated and control groups.
• Discontinuous data was subjected to non-parametric tests, for two groups Mann-Whitney U-Test and for more than two groups kruskal-wallis ANOVA was used.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In the satellite group of females on days 8, 36, 43, 50, 57 and 64 significant decrease in body weight gain (%) noticed in high dose group (G4S- 1000 mg/kg body weight) when compared to control (G1- 0 mg/kg body weight). Whereas no significant difference in the body weight gain (%) was observed in male animals compared to control group of animals.
The significant changes observed in body weight and body weight gain (%) are marginal and could not be attributed to test item administration.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Decrease in feed consumption of female intermediate (G3- 300 mg/kg body weight) dose group was observed as compared to control and low dose group on day 8 of treatment period.
During gestation period day 0 and 7 females showed significant decrease in feed consumption in high dose main group (G4- 1000 mg/kg body weight) when compared with control dose (G1-0 mg/kg body weight) groups.
The significant changes were observed in feed consumption in intermediate (G3-300 mg/kg body weight) and high dose (G4-1000 mg/kg body weight) groups are marginal, not dose dependent and could not be attributed to test item administration

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):

In Males during treatment period, increase in Neutrophils and decrease in MCV were observed in intermediate dose (G3-300 mg/kg body weight) and high dose (G4-1000 mg/kg body weight), respectively compared to control (G1- 0 mg/kg body weight).
In (G4S- 1000 mg/kg body weight) satellite group females a significant decrease in, Hb, PCV, MCV, MCH, Basophils and increase in monocytes were observed compared to control group (G1- 0 mg/kg body weight) of animals.
The significant differences observed in hematology parameters could not be attributed to treatment due to marginal increase or decrease in individual control values and in the absence of any biological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In Males during the treatment period a significant increase in AST and Potassium were observed in high dose (G4-1000 mg/kg body weight) compared to low dose group (G1- 0 mg/kg body) and control group (G1- 0 mg/kg body), respectively.
In high dose females (G4-1000 mg/kg body weight), a significance increase in AST compared to intermediate dose (G3-300 mg/kg body weight).
In satellite males, increase in AST was observed in high dose (G4S-1000 mg/kg body weight) compared to control group of animals.
Whereas in satellite females s significant increase in AST was observed at the high dose compared to control satellite group (G1S- 0 mg/kg body)
The significanct changes observed in biochemical parameters are non dose dependent and not biological significant, hence could not be attributed to treatment related adverse effect.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A significant increase in absolute and relative weight of liver was observed in male animals in high dose (1000 mg/kg) group when compared with control (0 mg/kg).
In female animals, significant increase in absolute and relative weight of liver was observed in high dose (1000 mg/kg) groups when compared with control (0 mg/kg)
A significant increase in absolute and relative weight of liver was observed in satellite group male and female animals in high dose satellite (1000 mg/kg) group when compared with control satellite (0 mg/kg) group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Necropsy performed at the end of the treatment period revealed test item related pale liver in 11/12 males and 12/12 females of high dose (1000 mg/kg) group; 5/12 males and 4/12 females of intermediate dose (300 mg/kg) group. Following recovery period, no abnormality was observed in male and female animals of control satellite (0 mg/kg) and high dose satellite (1000 mg/kg) groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination revealed vacuolation in liver of 12/12 male and 12/12 female animals of high dose (1000 mg/kg) and 12/12 male and 9/12 female animals of intermediate dose (300 mg/kg) group. Microscopically, high dose satellite (1000 mg/kg) group revealed vacuolation in liver of 5/5 male and 5/5 female animals after a treatment free 14-day recovery period.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Applicant's summary and conclusion

Conclusions:

The administration of LICOWAX R 21 S FL to Wistar rats by oral gavage, at dose levels of 0, 100, 300 and 1000 mg/kg/day, resulted in no treatment-related clinical signs up to the high dose group. Changes in the body weights, feed consumption and hepatocytes were observed at 1000 mg/kg bw/d. No treatment-related effects on reproduction/ development such as mating index, fertility index, gestation length, pre-natal loss, post-natal loss, sex ratio and offspring growth and development were reported. Under the condition of this study vacuolation in liver was recorded at 1000 and 300 mg/kg bw/d (at 300 mg/kg bw/d not additinal changes were observed) in both sexes. These changes persisted after a treatment free period of 14 days at the 1000 mg/kg body weight satellite group.
Therefore, a precautionary ‘No Observed Effect Level’ (NOAEL)` was derived at 300 mg/kg bw/d.

Executive summary:

The test item, Licowax R 21 S FL (formulated in Refined Ground Nut Oil) was administered by gavage to three treatment groups, each oftwelve male and twelve female Wistar rats, for up to 42 days. Males were treated for two weeks premating phase, up to two weeks mating and post-mating, females for two weeks premating phase, up to two weeks mating, three weeks gestation and 13 days lactation at dose levels of 100, 300 and 1000 mg/kg bw/d. A control group of twelve males and twelve females was dosed with the vehicle control (refined ground nut oil). A satellite group of five males and five females was dosed at 1000 mg/kg bw/d and a satellite control group of five males and five females was dosed with the vehicle for a preiod of 50 days followed by 14 days treatment free period.

The following observation and examinations were evaluated:

- Clinical signs (daily), body weights and feed consumption (once weekly, except during mating).

- Pairing of 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.

- Five male and five female animals selected for repeated dose toxicity screening and all the animals of satellite groups.

- Macroscopy at termination was performed for all animals.

- All males and females were evaluated for Hematology, Clinical biochemistry, Urinalysis at the end of treatment period.

- Male and female animals from each dose group were screened for T4 and TSH parameters. The offspring selected from each dose group was also screened for T4 and TSH.

- Testes, epididymides, seminal vesicle + prostate of all adult males; uterus, ovaries of all female animals and following organ weights were recorded on scheduled dates of necropsy from five male and five female animals selected for repeated dose toxicity screening and all the animals of satellite groups:

Liver spleen, kidneys, brain, adrenals, heart and thymus

- From all adult males and females and two pups from day 13 post natal from each litter, thyroid weight was determined after fixation.

- Histological preparation of the tissues was performed of all organs and tissues from all animals of the control (0 mg/kg) and high dose (1000 mg/kg) were processed and examined microscopically

Results:

- Analysis of stability, homogeneity and dose concentration of the prepared dose formulations revealed acceptable levels.

- There were no mortalities in male and female animals observed

- No clinical signs were observed

- During clinical observation no abnormalities were observed in all the animals

- Functional observation battery parameters like grip strength (Forelimbs and Hind limbs), Rotarod and IR Actimeter did not show any treatment related significant changes in all the treated groups when compared with control group

- The significance observed in body weight and body weight gain (%) in high dose (G4 and G4S-1000 mg/kg body weight) are marginal and could not be attributed to test item administration.

- no test item related changes in food consumption

- The significant differences observed in hematology and clinical biochemistry parameters could not be attributed to the treatment dur to marginal increase or decrease in individual control values and have no biological significance. However, there was an increase of the AST level noted in all high dose animals.

- No treatment related significant differences were observed in urinalysis parameters

- No toxicologically relevant findings were noted in thyroxine (T4) and thyroid stimulating hormone (TSH) levels in parent animals (males, females) and pups (male and female).

- No test item related change was observed in organ weights without one exception: an increase in absolute and relative liver weight was observed in male high dose animals. In female animlas a increase in absolute and relative liver weight in male and female animals of the high dose satellite group was reported.

- Necropsy performed at the end of the treatment period revealed test item related pale liver in 11/12 males and 12/12 females of high dose (1000 mg/kg) group; 5/12 males and 4/12 females of intermediate dose (300 mg/kg) group. Following recovery period, no abnormality was observed in male and female animals of control satellite (0 mg/kg) and high dose satellite (1000 mg/kg) groups.

- Microscopic examination revealed vacuolation in liver of 12/12 male and 12/12 female animals of high dose (1000 mg/kg) and 12/12 male and 9/12 female animals of intermediate dose (300 mg/kg) group. No abnormality attributable to the test item was observed in low dose (100 mg/kg) group animals. Microscopically, high dose satellite (1000 mg/kg) group revealed vacuolation in liver of 5/5 male and 5/5 female animals after a treatment free 14-day recovery period.

The test item eclusively consists of tallow fatty acid components and therefore, can be regarded as animal fat. In case of an excessive supply of high-energy food, like the uptake of animal fat, mammalian organisms do not excrete the excess energy carrier but "store" it in the tissue, mainly in the liver. This mechanism might lead to the incorporation of fatty acids in hepatocytes in form of vacuolation. The so called fatty metamorphosis of the liver in one of the most frequent changes in humans (ca. 25% of all adults) and if no additional changes, i.e. inflammation of the tissue, can be diagnosed, this indication is regarded as non-relevant in medicine.

Since the test item is a mixture of animal fatty acids the observed vacuolation in hepatocytes is considered to be the result of an unbalanced diet and the vacoules are assumed to contain excess fatty acids, which are commonly stored in the liver. No additional effects, like the increase of inflammatory markers in blood or inflammatory histopathological changes, increase in enzyms in blood (i.e. aminotransferases as indicator of cellular decline), clinical symptoms, changes in organ weights or affected reproductive performance are observed up to 300 mg/kg bw/d. Therefore, the occurrence of fatty vacuoles in the liver should be judged as non-adverse (adaptive) effect and the NOAEL is derived at 300 mg/kg bw/d (at 1000 mg/kg bw/d an unspecific increase of AST lacking dose response relationship is observed in all animals) as most conservative approach.