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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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

Justification for study design:

n.a.

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 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

Details on mating procedure:

- M/F ratio per cage: one male : one female
- Length of cohabitation: max. 18 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually

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

Details on study schedule:

1000 mg/kg bw/d

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

Parental animals: Observations and examinations:

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)

Oestrous cyclicity (parental animals):

All the females were screened 14 days for estrous cyclicity before start of the treatment

Sperm parameters (parental animals):

Two slides of testes section were taken at the time of sectioning. One slide was used for H & E staining for routine evaluation and second slide for Periodic acid-Schiff’s-Hematoxylin (PAS-H) staining for staging of spermatogenic cycle

Litter observations:

For each litter the following parameters were recorded:
i) Number of offspring born
ii) Number of offspring alive was recorded daily and reported on days 1 and 4 post partum
iii) Sex of offspring on day 1 post partum
iv) Clinical condition of offspring from birth to day 13 post partum
v) Individual offspring body weights on Days 1, 4 and day 13 post partum
vi) The Anogenital distance of each pup was measured on day 4 post-partum. The anogenital distance was measured from the cranial edge of the anus to the base of genital tubercle.
vii) The number of nipples in male pups was counted on day 13 post-partum
viii) Gross anomalies
Blood samples were collected for thyroid hormones analysis (T4 and TSH) as per the following schedule:
• Two pups per litter on day 4 after birth

Postmortem examinations (parental animals):

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.
The uteri of females were examined for the presence and number of implantation sites and the number of corpora lutea in the ovaries was determined.

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

Females which failed to achieve pregnancy were sacrificed after Day 26 post coitum. The uterus that appeared non-gravid and also gravid was stained with 5% ammonium sulphide solution to reveal any early resorption or post implantation loss.

Postmortem examinations (offspring):

Dead pups were subjected to gross examination, whereas live pups were sacrificed at day 13 post partum and gross examination was performed.

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.

Reproductive indices:

Mating Index (%)
(Number of animals mated/ Number of animals paired) x 100
Pregnancy Index (%)
(Number of pregnant females/ Number of female animals mated) x 100
Parturition Index (%)
(Number of females delivering live offspring/ Number of pregnant females) x 100
Pre–natal loss
Implantations - live births

Offspring viability indices:

Post– natal loss
Live births minus alive at post natal Day 4/13
Sex Ratio (% males)
Number of male offspring (Day 1 or Day 4) / Total number of offspring (Day 1 or Day 4) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 administrat

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

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P0)

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: no effect on fertility/reproduction observed

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Of the litters delivered, in all the treatment groups, litter size at birth and subsequently on Day 1 and 4 post-partum were comparable to controls. No significant difference in the live birth was noted between control and treated groups. Whereas marginal increase and decrease in percentage of live foetus was noticed low dose (G2- 300 mg/kg body) and high dose (G4- 1000 mg/kg body) compared to control group (G1- 0 mg/kg body) lacking significance.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No significant changes were observed in T4 and TSH levels of male and female offspring on day 4 and day 13 lactation periods in all the groups compared to control group (G1- 0 mg/kg body) of animals.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No significant differences in the Anogenital distances (AGD) between control and treated animals were observed.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect on development observed

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

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, changes in the body weights, feed consumption zp to the high dose group. 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. Therefore the NOEAL(reproduction/developmental) was derived at 1000 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).

The following observations 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.

- 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 were examined

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

Results:

1. Mating and fertility

No treatment-related effects were detected on mating performance. All the female animals of control, low, intermediate and high dose groups showed positive evidence of mating. No significant difference was observed in the pre-coital interval of female animals between control, low, intermediate and high dose group. Oestrous cyclicity was checked for all the female animals. Regular oestrous cycle was observed in all the animals of different groups in the study. Mating index for all groups was 100%.

One dam each from control, intermediate and high dose group was observed to be not pregnant. The pregnancy index for Control -0, Low-100, Intermediate-300 and high dose -1000 mg/kg/day groups were observed 100%.

Of the litters born, litter sizes at birth and subsequently on days 1 and 4 post partum were comparable to control

There were no treatment-related effects observed in fertility of treated animals.

2. Gestation and parturition

There were no differences in gestation lengths. The gestation length for treated females was comparable to controls. All animals showed gestation lengths between 20 to 24 days.

The parturition index was 91.67 % in control; intermediate dose and high dose groupwhereas in intermediate dose it was observed 100.00 %.

3. Pre and post natal loss

The pre and post natal losses in all treated groups were comparable with findings in the control group whereas marginal decrease and increase in percentage post implantation loss was observed in the low dose group and high dose group, respectively. No dose response relationship was observed.

4. Litter size and live birth

Of the litters delivered, in all the treatment groups, litter size at birth and subsequently on Day 1 and 4 post-partum were comparable to controls. No significant difference in the live birth was noted between control and treated groups. No nipple retention was found in any of the male litter from vehicle control, low, intermediate and high dose groups.

5. Sex ratio

No significant difference in sex ratio was observed in any of the treated groups when compared with vehicle control.

6. Offspring growth and development

Offspring body weights were recorded on day 1, day 4 and 13 were recorded. No significant changes in the offspring body weight were observed in Low (G2- 100 mg/kg body), intermediate (G3- 300 mg/kg body) and high dose group (G4- 1000 mg/kg body) when compared with control (G1- 0 mg/kg body) group.

No significant differences in theAnogenital distances (AGD) between control and treated animals were observed

7. Litter weights

Offspring body weights on day 1, day 4 and 13 were recorded. No significant changes in the offspring body weight were observed.

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).

Based on the results of the OECD 422 study performed with Liccowax R21 S the NOAEL (reproduction/developmental) is derived at 1000 mg/kg bw/d.