Fatty acids, vegetable-oil, Me esters, sulfurized

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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)

Version / remarks:

OECD Guideline no. 422 adopted on 29 July 2016

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 was the species and strain of choice because it is accepted by many regulatory authorities and because there are 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

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

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on exposure:

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

Details on mating procedure:

- M/F ratio per cage: one male to one female
- Length of cohabitation: until positive identification of copulation occurred or 14 days had elapsed
- Proof of copulation: Vaginal smears were taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plug found on the cage tray).
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: 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.
- Any other deviations from standard protocol: Animals were ordered with a body weight range of approximately 200 to 225 g for males and 175 to 200 g for females but the weight at arrival was between 220-230 g for males and 183-191 g for females.

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:

Males were dosed for 2 consecutive weeks prior to pairing and up to the day before necropsy, for a total of 29/30 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females were dosed for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifice, for a total of 51-64 days.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.

Frequency of treatment:

Once a day, 7 days a week.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on information from a preliminary, non-GLP compliant study (RTC Study No. E0240).
- Fasting period before blood sampling for clinical biochemistry: not stated

Positive control:

No positive control

Examinations

Parental animals: Observations and examinations:

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.

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.
- Cage side and detailed clinical observations checked are stated in “TABLE 3 - Clinical observation” of the attached final report no. X0910.

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.

Oestrous cyclicity (parental animals):

Stock females
Oestrus cycle was monitored by vaginal smears in all stock females for at least 2 weeks before allocation in order to exclude from the study females with irregular cycle.

Females allocated to groups
Vaginal smears were taken in the morning from Day 1 of dosing up to positive identification of mating. The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)

Vaginal smears were also taken from all females, before despatch to necropsy.

Sperm parameters (parental animals):

Parameters examined in male parental generation: A detailed qualitative examination of the testes was performed in all control and high dose group males killed at term. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952 and referred to the comprehensive reviews on the subject Russell, 1990; Creasy, 1997; Creasy, 2002. The PAS-H stained sections were used to identify the spermatogenic stages.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages. Testis weight and epididymis weight was examined as well.

Litter observations:

STANDARDISATION OF LITTERS
On Day 4 post partum, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment (for example, 3 males and 5 females) was acceptable.
Two culled pups (one male and one female where possible) were selected for hormone determination. If litters did not have extra pups to be culled (i.e.: litters with 8 pups or less), at least 1 female pup was sacrificed for hormone determination in order to retain more male pups for nipple retention on Day 14 post partum. However, retained female pups in each litter were not below 2.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
As soon as possible after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1, 4 and 13 post partum. Observations were performed once daily for all litters. Pups killed or dying during the lactation period were weighed before the despatch to necropsy.
After culling all pups were sacrificed with the dams on Day 14 post partum.

GROSS EXAMINATION OF DEAD PUPS:
Yes, all pups found dead in the cage were examined for external and internal abnormalities. The possible cause of death was not determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not examined
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not examined

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after 29/30 days
- Maternal animals: All surviving animals were sacrificed after 51-64 days.

GROSS NECROPSY
- Gross necropsy consisted of an 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. Details are stated in “APPENDIX 25 -Macroscopic and microscopic observations - Individual data” of the attached final report no. X0910.

HISTOPATHOLOGY / ORGAN WEIGHTS
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). Details are stated in “APPENDIX 23 - Absolute organ weight - Individual data” and in “APPENDIX 25 - Macroscopic and microscopic observations - Individual data” of the attached final report no. X0910.

Postmortem examinations (offspring):

SACRIFICE
- Pups were euthanized by intraperitoneal injection of Thiopental on Day 4 or Day 14 post partum.
- 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. Additionally the Thyroid weight for the pups sacrificed at Day 14 post partum was determined.

GROSS NECROPSY
- Gross necropsy consisted of external examinations and sex determined by internal gonads inspection. Detailed results are stated in “APPENDIX 21 - Necropsy findings in pups - Individual data” of the attached final report no. X0910.

ORGAN WEIGTHS
The Thyroid weight for the pups sacrificed at Day 14 post partum are stated in “APPENDIX 22 - Pups thyroid weight on Day 14 post partum - Individual data” of the attached final report no. X0910.

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 the Williams test. The criterion for statistical significance was p<0.05.

Reproductive indices:

The copulatory and fertility Index was calculated for males and females as follows:

Males
-----------------------------------------------------------------------------------------
Group 1 2 3 4
-----------------------------------------------------------------------------------------
Copulatory Index% 100.0 100.0 100.0 100.0
-----------------------------------------------------------------------------------------
Fertility Index% 100.0 100.0 100.0 100.0
-----------------------------------------------------------------------------------------
Copulatory Index (%) = No. of animals mated / No. of animals paired x 100
Fertility Index (%) = No. of animals which induced pregnancy / No. of animals paired x 100


Females
-----------------------------------------------------------------------------------------
Group 1 2 3 4
-----------------------------------------------------------------------------------------
Copulatory Index% 100.0 100.0 100.0 100.0
-----------------------------------------------------------------------------------------
Fertility Index% 100.0 100.0 100.0 100.0
-----------------------------------------------------------------------------------------
Copulatory Index (%) = No. of animals mated / No. of animals paired x 100
Fertility Index (%) = No. of pregnant females / No. of females paired x 100

Offspring viability indices:

not stated

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs occurred during the study. The sporadic clinical signs, such as hairloss, piloerection and scabs, detected in few treated animals, were considered to be incidental.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in the study. All females were pregnant and with live pups on Day 14
post partum.

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 examined

Description (incidence and severity):

not applicable

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The sporadic lesions reported in control and/or treated animals such as retinal dysplasia of the eye in one high dose (1000 mg/kg/day) male and female (nos. X0910064 and X0910061), pelvic dilatation of kidneys in two high dose (1000 mg/kg/day) males (nos. X0910062 and X0910064) and one low dose (100 mg/kg/day) female (no. X0910021), adenoma of mammary gland and necrosis of liver in one mid-dose (300 mg/kg/day) female (nos. X0910051 and X0910041, respectively) were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cycle, pre-coital intervals, copulatory index and fertility index did not show any treatment-related intergroup differences.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No treatment-related changes were noted. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Reproductive performance:

no effects observed

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

haematology

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related effects. Pre-weaning clinical signs were comparable between treated and control groups.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Description (incidence and severity):

not applicable

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

not applicable

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Group 4 (1000 mg/kg/day) males showed low thriiodothyronine values. However, similar data were recorded in all groups, including controls, therefore this finding was considered to be unrelated to treatment. One female pup of Group 3 [(dam no. X0910053) 300 mg/kg/day] showed high thriiodothyronine. Due to the absence of dose-relation, this change was considered to be incidental.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Mean anogenital distance (normalised value), performed on Day 1 post partum, showed statistically significant increase in mid- and high dose groups, both for male and female pups, compared to controls. Due to the absence of dose-relation, these changes were considered to be of no toxicological relevance.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples were observed in male pups.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant decrease (approximately 18%) was recorded in thyroid weight of male pups on Day 14 post partum, when compared to controls.
Due to the absence of dose-relation, this change was considered to be of no toxicological relevance.
No significant differences were observed in the thyroid weight of female pups, when compared to controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect.

Histopathological findings:

not examined

Other effects:

not specified

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

clinical biochemistry

gross pathology

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

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.