Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 October to 11 January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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 (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Fatty acids, C18-unsatd., dimers, oligomeric reaction products with 1-chloro-2,3-epoxypropane
EC Number:
500-215-4
EC Name:
Fatty acids, C18-unsatd., dimers, oligomeric reaction products with 1-chloro-2,3-epoxypropane
Cas Number:
68475-94-5
Molecular formula:
Not applicable for a UVCB substance please see remarks section below.
IUPAC Name:
Fatty acids, C18-unsatd., dimers, oligomeric reaction products with 1-chloro-2,3-epoxypropane
Test material form:
liquid
Specific details on test material used for the study:
Identification: Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin
CAS Number: 68475-94-5
Physical state/Appearance: Clear liquid
Batch: 52611021
Purity: 100% (UVCB product)
Expiry Date: 1 Dec 2018
Storage Conditions: Room temperature in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 15 to 16 weeks
- Weight at study initiation: Males: 449 - 643 grams, Females: 233 - 328 grams
- Housing: Polycarbonate cages with a stainless steel mesh lid.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITION
- Temperature (°C): 20 to 26
- Humidity (%): 30 to 70
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Fatty Acids, C18-Unsatd., Dimers, Polymers with Epichlorohydrin was prepared for administration by mixing appropriate amounts of the test item with the vehicle to achieve the desired concentrations. The test item was used as supplied when calculating quantities to be used during dose preparation. Fresh formulations were prepared once weekly and were stored frozen (-20 °C) when not in use. Detailed records of test item usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined at each preparation.
VEHICLE
- Concentration in vehicle: 0, 10, 30, 100mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no.: 2GH0193, 2GK0223
- Purity: Assume 100 %
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity:
Homogeneity of the dose formulation was demonstrated by taking two samples each (2.5 mL/sample) from the top, middle and bottom portion of the low and high concentrations prepared for use on the study.
Stability:
Stability under storage conditions used in this study (24 hours at room temperature; 8 days refrigerated [2 to 8°C] and up to 9 months frozen [-20°C ± 10ºC]); were performed. Five (5) grams of the test item and 200 mL of the vehicle were sent to the Test Site for stability assessment.
Dose Concentration:
Two samples (2.5 mL each) were taken from the middle region of each formulation (including control) for each week on the day of dose preparation. For the first and seventh dose formulations prepared, one sample was analyzed, in duplicate, for dose confirmation analysis and one sample was retained refrigerated (retained sample was discarded after valid analytical results were obtained).
Storage:
All dose formulation samples were stored frozen (-20°C ± 10ºC) until shipped to the Test Site for analysis.
Duration of treatment / exposure:
The duration of dosing for the main study F0 males was 2 weeks pre-cohabitation, during cohabitation (up to 3 weeks) and continuing during post-cohabitation until the day prior to termination [approximately 35 days].
The duration of dosing for the main study F0 females was 2 weeks pre-cohabitation, cohabitation (up to 3 weeks) and during gestation and lactation continuing until LD 13 (maximum of 60 days).
Frequency of treatment:
Rats were dosed once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
control group
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 male and 10 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previous 14-day oral dose study in rats at initial doses of 100 and 1000 mg/kg/day, there was an average decreased weight gain for the males and slight weight loss for the females dosed at 1000 mg/kg/day on Day 2. After 4 days of dosing at 1000 mg/kg/day, two of six animals (1 male and 1 female) were in poor condition that included decreased activity, irregular breathing and irregular gait, and were subsequently euthanized. The remaining 2 per sex dosed at 1000 mg/kg/day were in normal health and were given a “dose holiday”. Of the remaining 2 per sex that were dosed at 1000 mg/kg/day, 2 males and 1 female were considered in good health and after 3 days of “dose holiday” resumed dosing for 7 days at 500 mg/kg/day. The animals dosed for 7 days at 500 mg/kg/day and the animals dosed for 14 days at 100 mg/kg/day were in good health at the end dosing. Based on the results of the previous study, the dose levels for this study are 50, 150 and 500 mg/k/day.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: Yes K2EDTA
- Animals fasted: Yes
- How many animals: All F0 rats
- Parameters checked in table [No.?] were examined. Hemoglobin (HGB)
Hematocrit (HCT)
Red blood cell count (RBC)
Platelet count (PLT)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Red cell distribution width (RDW)
Total white blood cell count (WBC)
Reticulocyte count (RETIC)
Differential white blood cell count1
Neutrophils (ANEU)
Lymphocytes (ALYM)
Eosinophils (AEOS)
Basophils (ABASO)
Monocytes (AMONO)
Large unstained cells (ALUC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination
- Animals fasted: Yes
- How many animals:
- Parameters checked in table [No.?] were examined. Aspartate aminotransferase (AST) (Kinetic - Modified Bergmeyer)
Alanine aminotransferase (ALT) (Kinetic - Modified Bergmeyer)
Alkaline phosphatase (ALKP) (Kinetic – Tietz AMP Buffer)
Blood urea nitrogen (BUN) (Enzymatic – Roch-Ramek with Urease)
Creatinine (CREAT) (Jaffe Picric Acid in Alkaline Medium)
Glucose (GLU) (Glucose Hexokinase II Method)
Cholesterol (CHOL) (Enzymatic esterase/oxidase – Trinder Endpoint)
Triglycerides (TRIG) (Fossati Three Step Enzymatic – Trinder Endpoint)
Total protein (TP) (Biuret Technique)
Albumin (ALB) (Bromocresol Green Method)
Total bilirubin (TBILI) (Oxidation with Vandate)
Sodium (NA+) (Ion Selective Electrode)
Potassium (K+) (Ion Selective Electrode)
Chloride (Cl-) (Ion Selective Electrode)
Calcium (Ca++) (Michaylova & Ilkova, Arsenazo III)
Inorganic phosphorus (PHOS) (Phosphomolybdate - UV Method

URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Thyroid Hormone Analysis – F0 Animals and F1 Pups

F0 males and females at termination - Up to 10 animals/sex/group
F pups at PND 4 - Up to 2 females per litter (when possible) pooled sample by litter
F pups at PND 13 - Up to 2 males and 2 females (when possible) itter
Sacrifice and pathology:
Sacrifice:
- Male animals: Necropsy was performed on 10 F0 males/group after males had been treated for 5
weeks.
- Maternal animals: Necropsy was performed on up to 10 F0 females in Groups 1 and 2 on LD 14.


GROSS PATHOLOGY: Yes (see table) / No / Not specified

HISTOPATHOLOGY: Yes (see table) / No / Not specified
Statistics:
All statistical analyses were carried out using the individual animal (or litter) as the basic experimental unit except where noted below.
For litter findings, the litter was the treated experimental unit and the basis for statistical analysis and biological significance were assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
Animals that were not pregnant were excluded from group mean body weight and food consumption calculations.
The following comparisons were made:
Group 1 vs 2, 3 and 4
For continuous parameters:
For comparisons involving more than two groups, if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level, then parametric methods were applied. If the F1 approximate test for monotonicity (described below) of dose response was not significant at the 1% level, two-tailed Williams' test (Williams 1971, 1972) for a monotonic trend was applied. If the F1 test was significant, suggesting that the dose-response was not monotone, then two-tailed Dunnett's test (Dunnett 1955, 1964) was performed instead. Dependant on the results a two-tailed Shirley's test for a monotonic trend (Shirley 1977) was applied. If the H1 test was significant, then two-tailed Steel's test (Steel 1959) was performed instead.

For discrete parameters:
For comparisons involving more than two groups either the two-tailed Jonckheere-Terpstra test, one-tailed step-down testing or the the Kruskal-Wallis test was applied. If the Kruskal-Wallis test was significant at the 5% level, the test-item treated groups
were compared to the Control using exact two-tailed Wilcoxon rank sum tests; otherwise, no further comparisons were made. Comparisons involving only two groups were made using two-tailed Wilcoxon rank sum tests.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were test item-related clinical observations during postmating for males and gestation for females at 500 mg/kg/day Fatty Acids, C18-Unsatd., Dimers, Polymers with Epichlorohydrin.
Similar clinical signs (abnormal gait and flattened posture) were noted for both sexes. However, these signs presented earlier in the majority of the males (Postmating Days 6 - 8) compared to the females (Gestation Days 23 – 25).
There were no test item-related clinical observations during the following phases: precohabitation (both sexes), cohabitation (both sexes), and lactation (females only).
Mortality:
no mortality observed
Description (incidence):
Three 50 mg/kg/day females (Animal Nos. 2532, 2536, 2540) and all ≥150 mg/kg/day females were electively euthanized prior to the scheduled termination date because they failed to litter. The deaths of these unscheduled decedents were not considered directly related to fatty acids, c18-unsatd., dimers, polymers with epichlorohydrin, however findings of epithelial hyperplasia/hypertrophy in the vagina and/or cervix/uterus were related to reproductive failure leading to euthanasia.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were test item-related decreases in body weight changes in males and females at 500 mg/kg/day and 150 mg/kg/day Fatty Acids, C18-Unsatd., Dimers, Polymers with Epichlorohydrin, respectively.
In the males, the decrease in body weight change (-0.44% of the respective control). was noted during the mating phase from Days 7 - 14. In the females, the maximal decrease of -78% of the respective control value was noted during the gestation phase for the GD 14 – 20 interval.
There were no test item-related decreases in body weight or body weight change at 50 mg/kg/day (both sexes) or 150 mg/kg/day (males) or 500 mg/kg/day (females).
Description (incidence and severity):
There were test item-related effects on male food consumption during the postmating phase in the 500 mg/kg/day Fatty Acids, C18-Unsatd., Dimers, Polymers with Epichlorohydrin group and on female food consumption during lactation in the 50 mg/kg/day Fatty Acids, C18-Unsatd., Dimers, Polymers with Epichlorohydrin group (the highest dose group that delivered).
Male food consumption was reduced (87.5% of the control group value) during the postmating phase in the 500 mg/kg/day Fatty Acids, C18-Unsatd., Dimers, Polymers with Epichlorohydrin group.
Food consumption during the gestation phase in the 150 mg/kg/day female group were slightly but not adversely reduced when compared with control group values. Food consumption during the lactation phase in the 50 mg/kg/day female group were significantly reduced on LDs 1 to 7 and LDs 7 to 14 when compared with control group values. The 150 and 500 mg/kg/day Fatty Acids, C18-Unsatd., Dimers, Polymers with Epichlorohydrin female group food consumption
could not be evaluated during the lactation phases due to none of the females having delivered.
Food consumption in the 50, 150 and 500 Fatty Acids, C18-Unsatd., Dimers, Polymers with Epichlorohydrin male and female groups were comparable with control group values during Precohabitation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related clinical chemistry changes at 50 or 150 mg/kg/day. Clinical chemistry changes at 500 mg/kg/day in males only included increases in alanine aminotransferase activity (1.53× control); and increases in albumin (1.08× control), total protein (1.07× control), and calcium (1.07× control).
Animal No. 2022 had mildly higher alanine and aspartate aminotransferase values. These higher values were considered not test item related as they lacked a dose relationship (correlates).
These changes were considered nonadverse due to their relatively small magnitude.
All other differences between control and treated mean values, including those that were statistically significant, were considered not test item related as they lacked a dose relationship and/or there was general overlap between individual control and treated values.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no effects on the FOB for the males (Week 5) ) at ≤ 500 mg/kg/day Fatty Acids, C18-Unsatd., Dimers, Polymers with Epichlorohydrin and for the females (LD 8) ) at 50 mg/kg/day Fatty Acids, C18-Unsatd., Dimers, Polymers with Epichlorohydrin.
Grip Strength
Males
There were test item-related statistically significant reductions in hindlimb grip strength (68% of the control group value) in the male (Week 5) 500 mg/kg/day Fatty Acids, C18-Unsatd., Dimers, Polymers with Epichlorohydrin group. There were non-adverse reductions in hindlimb grip strength in the male 50 and 100 mg/kg/day Fatty Acids, C18-Unsatd., Dimers, Polymers with Epichlorohydrin groups. The reductions were 74.9% and 72.6% in the respective groups. Due to
limited number of animals and the inherent variability in this parameter, the statistically significant value was considered non-adverse.
There was a non-adverse reduction in forelimb grip strength (89.8% of the control group value) in the male (Week 5) 500 mg/kg/day Fatty Acids, C18-Unsatd., Dimers, Polymers with Epichlorohydrin group. There were slight non-adverse increases in hindlimb grip strength in the male 50 and 100 mg/kg/day Fatty Acids, C18-Unsatd., Dimers, Polymers with Epichlorohydrin groups.
Females
There were no test item-related effect in forelimb grip strength in the female (LD 8) 50 mg/kg/day Fatty Acids, C18-Unsatd., Dimers, Polymers with Epichlorohydrin group. There was a non-adverse reduction in hindlimb grip strength (88.0% of the control group value) in the female (LD 8) 50 mg/kg/day Fatty Acids, C18-Unsatd., Dimers, Polymers with Epichlorohydrin group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Higher absolute and relative liver weights were observed in 500 mg/kg/day males compared to controls. Higher liver weights correlated to centrilobular hypertrophy observed microscopically.
Lower absolute and relative prostate and epididymides weights were observed in 500 mg/kg/day males compared to controls. Lower prostate and epididymides weights correlated to prostatic and epidydimal atrophy observed microscopically. Lower seminal vesicle/coagulating gland and levator ani/bulbocavernosus muscle complex weights were also observed in 500 mg/kg/day males compared to controls. Lower seminal vesicle/coagulating gland weights correlated to
small seminal vesicles/coagulating glands observed macroscopically.
There were no test item-related changes in organ weights in females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
A small prostate was observed one 500 mg/kg/day male and correlated to prostatic atrophy microscopically.
Small horn(s) in seminal vesicles/coagulating glands were observed in three 500 mg/kg/day males.
All other macroscopic findings occurred sporadically or at similar incidence and severity in control and test item-treated groups and were considered incidental and due to biological variability.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic findings related to fatty acids, c18-unsatd., dimers, polymers with epichlorohydrin were observed in the uterus/cervix, and vagina at ≥50 mg/kg/day, prostate gland at ≥50 mg/kg/day, epididymides at 500 mg/kg/day, and liver at ≥50 mg/kg/day.
Female Reproductive Tract
Epithelial hyperplasia/hypertrophy (minimal) of the vagina and/or uterus/cervix was observed in female rats at ≥50 mg/kg/day. Epithelial hyperplasia/hypertrophy was characterized by increased numbers of luminal and glandular (uterus) cell layers. Epithelial hyperplasia/hypertrophy in the vagina sometimes occurred with mucification, in which epithelial cells were enlarged with mucin-filled vacuoles, and extracellular mucous within the vaginal lumen.
Male Reproductive Tract
Atrophy (minimal to slight) of the prostate was observed in male rats at 500 mg/kg/day. Atrophy was characterized by reduced acinar/vesicle lumina with decreased acinar secretory material and crowding of intra-acinar/vesicle epithelial folds. Atrophy of the epididymides was observed in male rats at 500 mg/kg/day. Epidydimal atrophy was characterized by smaller ductal lumina containing reduced sperm content and germ cells and/or cellular debris.
Liver
Centrilobular hepatocellular hypertrophy (minimal) was observed in the livers of males and females at ≥50 mg/kg/day. This finding was characterized by enlarged hepatocytes and compressed sinusoids surrounding centrilobular veins.
All microscopic findings, including a single instance of pyelonephritis in a 50 mg/kg/day male, occurred sporadically or at similar incidence and severity in control and test item treated groups and were considered incidental and due to biological variability. Rodents are prone to spontaneous cases of pyelonephritis due to low perfusion in the vasa recta, hypotonicityassociated depression of leukocytic phagocytic activity, and continuity with ureters and urinary bladder where bacteria may be harboured.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
LOAEL
Effect level:
>= 50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (actual dose received)
System:
female reproductive system
Organ:
cervix
uterus
vagina
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
dorsolateral prostate gland
ventral prostate gland
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

There appears to be a dose response relationship, although not specified in the report, in the effects seen in the uterus and cervix however the effects seen are classed as minimal.

Applicant's summary and conclusion

Conclusions:
Based on the findings at 50 mg/kg/day, a NOAEL could not be determined in this study.
Executive summary:

The study was performed to assess possible effects of Fatty Acids, C18-Unsatd., Dimers, Polymers with Epichlorohydrin on male and female reproductive performance, including a repeated dosing toxicity part (in accordance with Test Guideline 422) when the test item is administered by oral gavage. This study allowed for the detection of effects on gonadal function, mating behavior, conception, development of the conceptus, parturition and pup survival to Postnatal Day 13 (PND 13).

The F0 males were dosed during precohabitation, cohabitation and post-mating periods (approximately 35 days) and then euthanized and necropsied. The F0 females were dosed during the pre-cohabitation and cohabitation periods and during gestation and lactation through Lactation Day 13 for approximately 63 days and then euthanized and necropsied. Parameters evaluated during the study for the F0 animals were:

viability, clinical observations, body weights, food consumption, estrous cycling, mating and fertility, parturition and littering, functional assessments, clinical pathology (termination), hormone analysis, organ weights, macroscopic observations and microscopic pathology. Parameters evaluated for the F1 pups were: viability, clinical observations, sex, body weights, ano-genital distance, nipple retention, hormone analysis, macroscopic observations and microscopic examination of the thyroid gland.

There were test-item related clinical observations noted, changes in bodyweight, effects on food consumption, altertions to estrous cycle, reductions in pregnancy and both male and female fertility indices, reductions in litter viability and both macroscopic and microscopic histopathological adverse effects.

In females and males, the administration of ≥50 mg/kg/day fatty acids, c18-unsatd., dimers, polymers with epichlorohydrin was considered adverse due to microscopic findings of epithelial hyperplasia/hypertrophy in the uterus/cervix and vagina associated with failure to litter in females and prostatic atrophy in males at this dose.

In conclusion, based on the findings at 50 mg/kg/day, a NOAEL could not be determined in the current study.