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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In the absence of data on fertility on target substance Fatty acids, C14-22, C16-24-alkyl esters an analogue read-across approach was conducted on suitable source substances:

Oral: Screening on toxicity to reproduction (OECD 422): NOAEL fertility (male/female) ≥ 1000 mg/kg bw/day

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Dec 2012 - 29 Jan 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Wistar Hannover RccHan
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Models, S.L., Barcelona, Spain
- Age at study initiation: 11-12 weeks
- Mean weight at study initiation: males: 312 to 364 g; females: 198 to 219 g
- Fasting period before study: no
- Housing: cages with standard, granulated, softwood Lignocel S8/15 bedding
Pretreatment period: 5 per cage
Mating period: 1 male and 1 female per cage
Postmating: single housing
- Diet: pelleted standard Harlan Teklad 2014C rat/mouse maintenance diet; ad libitum
- Water: tap water; ad libitum
(Analyses of diet and water was performed.)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 30-60
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: 1% methylcellulose+1% Tween 80 in aqueous solution
Details on exposure:
VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 18 h
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as day 0 postcoitum
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of dose formulation was determined in samples taken from the formulation to be administered to Groups 1 to 4 at the start and end of treatment by GC-FID analysis.
Duration of treatment / exposure:
Males: two weeks prior to mating and at least up to and including the day before sacrifice (day 49 of treatment).
Females: two weeks prior to mating and at least up to and including the day before sacrifice (day 4 postpartum).
Frequency of treatment:
Once daily; 7 days/week
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a dose-range finding toxicity study in rats (Harlan Laboratories Study S40412 14-day Oral (gavage) DRF using dose levels of 100, 500 and 1000 mg/kg bw/day.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 2 hours after dosing
Detailed clinical observations were performed on all test and control group animals before the first exposure to the test item and once weekly thereafter, at least two hours after dosing. Observations were also performed on females with litters on Day 4 postpartum. These observations were performed outside the home cage, in a standard arena, at least two hours after dosing (where applicable) to ensure that any transient effects of treatment are identified.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males: twice weekly during the pre-pairing and pairing period and daily during postpairing period.
F0 females: twice weekly during the pre-pairing and pairing period and daily until day 4-5 postpartum.

FOOD CONSUMPTION: Yes
F0 males: once weekly during the pre-pairing period and two weeks of postpairing period.
F0 females: once weekly during the pre-pairing period and days 0-7, 7-14, 14-21 postcoitum and days 1-4 postpartum.
No food consumption was recorded during the mating period.

WATER CONSUMPTION: Yes
F0 males: one week during the pre-pairing and postpairing period.
F0 females: one week during the pre-pairing, postcoitum period and days 1-4 postpartum.
No water consumption was recorded during the remaining periods.:
Oestrous cyclicity (parental animals):
Smearing of individual females was evaluated during pairing period and was discontinued when sperm are found.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight, qualitative staging of spermatogenesis and histopathology evaluation of interstitial cells of all males from the control and high-dose groups
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes (males on day 50, females on day 5 postpartum)
- Organ weights: epididymes and testicles, adrenal gland, brain, heart, kidney, liver, ovaries, pituitary, spleen, thymus, thyroid and parathyroids, uterus and oviducts
- Fixation: Adrenals, Aorta (thoracic), Bone with bone marrow- (Sternum), Peyer’s patches, Pharynx, Pituitary, Bone marrow smear (femur) (air-dried smear), Brain (cerebrum, cerebellum, medulla/pons), Epididymides (Bouin's solution), Esophagus, Eyes with optic nerve (Davidson’s fixative), Femur (with articular surface), Heart (with papillary muscle), Intestine, large (cecum, colon and rectum), Intestine, small (duodenum, jejunum and ileum), Kidneys, Larynx, Liver, Lungs, with main bronchi and bronchioles, Lymph nodes (mandibular and mesenteric), Nose (the entire head will be collected), Mammary gland area, Ovaries, Pancreas, Prostate, coagulating gland and seminal vesicles, Salivary glands (mandibular, sublingual and parotid), Sciatic nerve, Skeletal muscle, Skin (abdominal), Spinal cord (cervical, thoracic and lumbar), Spleen, Stomach (glandular and nonglandular), Testes (Bouin's solution), Thymus, Thyroid (incl. parathyroid gland, if possible), Tongue, Trachea, Urinary bladder, Uterus (cervix, corpus and oviducts), Vagina, All gross lesions
Postmortem examinations (offspring):
On day 4 postpartum, all live pups were sacrificed by an intraperitoneal injection of sodium pentobarbital and examined macroscopically.
Any F1 offspring that died during the study was examined externally and necropsied, except those excessively cannibalized. Samples of the organs and tissues with macroscopic alterations were taken and preserved in neutral phosphate buffered 4% formaldehyde solution for possible microscopic examination.
Statistics:
The following statistical methods were used to analyse food consumption during postpairing period, body weight, clinical laboratory data, organ weights, postnatal development and reproduction data, as well as macroscopic findings:
The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables can be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data cannot be assumed to follow a normal distribution.
Fisher's exact-test was applied to the macroscopic findings.
Armitage/Cochran Trend Test [5] for non-neoplastic lesions, if appropriate.
Bonferroni-test was applied to some reproduction parameters.
Reproductive indices:
- Percentage Mating [%] = Number of females mated/Number of females paired x 100
- Fertility Index female [%] = Females achieving pregnancy/Number of females paired x 100
- Gestation Index [%] = Number of litters with live pups/Number of pregnant rats x 100
- Concenption rate [%] = Females achieving pregnancy/Number of females mated x 100
- Pre-implantation loss [%] = Number of corpora lutea - Number of implantation sites/Number of corpora lutea x 100
- Post-implantation loss [%] = Number of implantation sites - Total number of offspring born/Number of implantation sites x 100
Offspring viability indices:
- Post-natal loss [%] = Number of offspring/Number of offspring alive on day 4 x 100
- Viability Index [%] = Number of offspring alive on day 4/Number of offspring alive on day 1 x 100

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No relevant clinical signs were observed. One female from the control group, one female and three males at 100 mg/kg bw showed occasionally up of the product (presumably immediate reaction to the test substance). In addition, one male at 300 mg/kg bw had missing upper incisors in week 4 of treatment. No clinical sings were observed at 1000 mg/kg bw in either males or females.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test-item-related differences from the control group were recorded. Some significant differences were observed at 1000 mg/kg bw/day but these were not considered of toxicological relevance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was similar in all groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
Slight increase in water consumption was recorded in females at 300 and 1000 mg/kg bw/day during pregnancy period. These differences were not statistically significant. In males no differences from the control group were recorded.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No changes of toxicological relevance were recorded.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No changes of toxicological relevance were recorded in females. Bilirubin, cholesterol and triglyceride values tended to be higher at 300 and 1000 mg/kg bw/day in females. An increase in bile acid values was observed at 300 mg/kg bw/day in males (non adverse). The differences recorded were statistically significant with respect to the control group.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Description (incidence and severity):
No differences in fore- or hind limb grip strength were recorded in males and females. Slightly lower locomotor activity was recorded in males at 100 mg/kg bw/day, being it statistically significant at 15 minutes. Slightly lower locomotor activity was recorded in females at 300 and 1000 mg/kg bw/day for 15-30 min, but the differences were not statistically significant. No differences were recorded in the remaining groups.

All animals responded positively to the different reflexes such as blink, pinna, righting and iridic reflexes and pain, auditory startle and handling response (push-off).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All microscopic findings recorded were considered to be within the range of normal background lesions that may be seen in rats of this strain and age and under the experimental conditions used in this study.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Female no.68 (1000 mg/kg bw/day) not mated with the first male (no. 28) after a pairing period of 14 days and mated a second time with male no. 23 showed an anestrus cycle of 11 days. Females nos. 43 (control group), 53 (100 mg/kg bw/day), 61 and 70 (300 mg/kg bw/day) and 73 (1000 mg/kg bw/day) took 12, 12, 13, 14 and 13 days, respectively, to mate and showed an anestrus cycle.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age. There were only single cases with isolated tubules that showed minor changes. They consisted of single sperm resorption in stage VII. In epididymides, there was only one animal in group 1 with unilateral monounclear cell foci of a minimal serverity.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating Performance: Median precoital time and mean precoital time (approx. 3 days) were similar in animals treated with the test item at 100 and 1000 mg/kg bw with respect to the control group. However, mean and median precoital time was longer at 300 mg/kg bw/day (5.7 and 4 days, respectively). In addition, female no. 68 from this group did not mate with the first male (no. 28) after a pairing period of 14 days and was mated a second time with male no. 23. This female showed an anestrus cycle of 11 days. Moreover, some animals from all groups took more than 2-4 days to mate. Females nos. 43 (control group), 53 (100 mg/kg), 61 and 70 (300 mg/kg bw/day) and 73 (1000 mg/kg bw/day) took 12, 12, 13, 14 and 13 days, respectively, to mate and showed an anestrus cycle.

Fertility: No treatment-related differences were recorded in the percentage of mating or the gestation index in females at the three doses with respect to the control group. Although fertility index and conception rate was lower in test-item-treated groups (100% vs. 90%), it was considered to be within the range of normal background findings which may be seen in rats.
Females no. 53 at 100 mg/kg bw/day, no. 70 at 300 mg/kg bw and no. 77 at 1000 mg/kg bw/day, which mated with males nos. 13, 30 and 37, respectively, were not pregnant despite the presence of sperm in the vaginal smear and vaginal plug in the last two females. Duration of the mating period of those couples was 12, 14 and 4 days, respectively. Females no. 69 at 300 mg/kg bw/day and no. 74 at 1000 mg/kg bw/day, which mated with males nos. 29 and 34, did not show signs of pregnancy and were mated again to ensure eight pregnant females per group. These animals were identified as 81 and 82 to record the data and their second mating led to pregnancy. To check the fertility of the male whose first pairing did not result in mating, male no. 28 from the 300-mg/kg bw/day group was mated a second time with one female from a reserve group and mating led to pregnancy.

Reproduction data:
No treatment-related differences from the control group were recorded in the mean of implantation sites per litter or corpora lutea.
No treatment-related differences from the control group were recorded in the percentage of pre- or postimplantation losses. Female no. 45 from control group showed 100% implantation site loss. No differences in sex ratio were recorded.

Breeding data:
The length of pregnancy was similar in all groups with a mean of 21-22 days.
Female no. 78 at 1000 mg/kg bw/day had not enough milk and did not nurse correctly its pups; consequently, the pups died or were devoured between days 2 and 3 of lactation. Despite the fact that the pups had milk in the stomach when alive, they did not have any at necropsy and showed hypothermia and pallor. Female no. 78 showed slightly enlarged mammary glands. Even the percentage of pups showing milk in the stomach was similar in all groups. One pup in the litter of females no. 43 from control group, no. 63 at 300 mg/kg bw/day, no. 78 and two pups from no. 73 at 1000 mg/kg bw/day showed no milk in stomach on day 1 postpartum and died.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed in the study
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed in the study
Key result
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No treatment-related differences from the control group were recorded in the mean of dead pups at the first litter check and living pups. During the first 4 days postpartum, the percentage and mean of postnatal losses was slightly higher at 1000 mg/kg bw/day; however the number of litters affected was similar to that of the control group (2 vs 1 litters) due to the fact that female no. 78 (1000 mg/kg bw/day) lost all its litter. The resulting viability index was lower than in the control group (84.7% versus 99.1%, respectively).
No differences were recorded at 100 and 300 mg/kg bw/day.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No differences were recorded between groups and sexes.
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:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No noteworthy findings were recorded in the control, 100, 300 or 1000 mg/kg bw/day pups.
No treatment-related alterations were recorded in the morphological examination of the pups. At 1000 mg/kg bw/day, one runt pup *no.7 from litter no.54) was observed.
One pup from control group had a hematoma on toes and one pup from 300 and 1000 mg/kg bw/day group had a wound on scapula or limbs.
Histopathological findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Motor development: Regarding postural reflexes in the pups, a significantly lower percentage of fetuses with positive response in the surface-righting reflex (righting reflex) at 1000 mg/kg bw/day was recorded compared to the control group. There were no treatment-related differences compared to the control animals at 100 or 300 mg/kg bw/day.
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
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 adverse effects observed in the study
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
06 Jun - 04 Aug 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 22 Mar 1996
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)BR
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
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: 191 to 204 g (males) and 172 to 179 g (females)
- Housing: From arrival to pairing: animals were housed 5 of one sex to a cage, in polysulphone solid bottomed cages measuring 59.5x38x20 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating: animals were housed one male to one female in clear polysulphone cages measuring approximately 43x27x18 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy). Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating; the females were transferred to individual solid bottomed cages (Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy) for the gestation period and parturition.
- Diet: laboratory rodent diet, 4 RF 21 (Mucedola S.r.l., Settimo Milanese (MI), Italy), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 2 weeks

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

Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous carboxymethylcellulose (0.5% CMC)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was suspended in the vehicle. Formulations were prepared daily.

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL for dose levels of 100, 300 and 1000 mg/kg bw/day, respectively
- Amount of vehicle: 10 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1 male to 1 female (monogamous).
- Length of cohabitation: The female was placed with the same male until pregnancy had occurred or 2 weeks had elapsed.
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- After 2 weeks of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged singly.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability of the test substance in the vehicle were verified by gas chromatopgraphy with a flame ionisation detection (FID). Concentration verification was conducted on a weekly basis.
Duration of treatment / exposure:
Males: The daily administration of the test item was started two weeks before mating and lasted until test day 28 to 29, which was one day before sacrifice.
Females: The daily administration of the test item was started two weeks before mating and continued until day 3 post-partum.
Maximum: 54 days of treatment.
Frequency of treatment:
once daily; 7 days/week
Details on study schedule:
not applicable for OECD 422 study
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a 14-day range-finding study (Rossiello, 2013. RTC Study No.: 93730EXT)
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily during the study, each animal was observed and any clinical signs recorded.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypes or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).

BODY WEIGHT: Yes
- Time schedule for examinations: females: weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

For further observations and examinations (water intake, haematology, clinical chemistry, neurobehaviour), see "Repeated dose toxicity: oral" (chapter 7.5.1)

OTHER:
Reproduction paramters: number of pregnant females, pre-coital time, gestation length
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning starting two weeks before pairing until a positive identification of copulation was made. The vaginal smear data were examined to determine the following: anomalies of the oestrous cycle and pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight, and qualitative sperm staging.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed. A detailed qualitative evaluation of testes was performed on 5 randomly selected control and high dose males. The evaluation took into account the tubular stages of the spermatogenic cycle, 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.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: litter weight, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [males were sacrificed on day 29 or 30]
- Maternal animals: All surviving animals [females were sacrifices on day 4 post-partum or shorty thereafter]

GROSS PATHOLOGY: Yes
-Organ weights: adrenal glands, brain (cerebrum, cerebellum, medulla/pons), epididymides, heart, kidneys, liver, ovaries with oviducts, parathyroid glands, prostate gland, seminal vesicles with coagulating glands, spleen, testes, thymus (where present), thyroid and uterus-cervix,
-Fixation: adrenal glands, bone marrow (from sternum), brain (cerebrum, cerebellum, medulla/pons), caecum, clitoral gland, colon, duodenum, epididymides, heart, ileum (including Peyer’s patches), jejunum, kidneys, liver, lungs (including mainstem bronchi), lymph nodes (mesenteric and cervical), ovaries with oviducts, parathyroid glands, pituitary gland, penis, preputial gland, prostate gland, rectum, sciatic nerve, seminal vesicles with coagulating glands, spinal column, spinal cord (cervical, thoracic, lumber), spleen, stomach, testes, thymus (where present), thyroid, trachea, urinary bladder, uterus-cervix and vagina.

HISTOPATHOLOGY: Yes, all organs that were included for fixation (5/sex of control and high dose group)
Postmortem examinations (offspring):
SACRIFICE
- The surviving F1 offspring were sacrificed at 4 days of age.


GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations].
Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.

HISTOPATHOLOGY / ORGAN WEIGTHS
not performed
Statistics:
Standard deviations were calculated as appropriate. For continuous variables 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. 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:
Male Copulatory Index (%) = No. of animals mated/No. of animals paired x 100
Male Fertility Index (%) = No. of males which induced pregnancy/ No. of males paired x 100
Female Copulatory Index (%) = No. of animals mated/No. of animals paired x 100
Female Fertility Index (%) = No. of pregnant females/No. of females paired x 100
Males and females:
Precoital interval = Mean number of days between pairing and mating
Offspring viability indices:
Pre-implantation loss [%] = (No. of corpora lutea - No. of implantations/ No. of corpora lutea) x 100
Pre-birth loss [%] = (No. of visible implantations - total litter size at birth/ No. of visible implantations
) x 100
Pup loss at birth [%] = (Total litter size - live litter size/ Total litter size) x 100
Cumulative pup loss on Day 4 post-partum [%] = (Total litter size at birth - live litter size at Day 4/ Total litter size at birth) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
No relevant clinical signs were observed in males and females throughout the study.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality was observed in males and females throughout the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No difference of toxicological significance were seen in body weight or body weight gain.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No intergroup differences were seen in food consumption.
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:
no effects observed
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):
No treatment-related changes were observed. The lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under the experimental conditions.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No relevant difference in oestrous cycle was observed in treated females when compared to controls.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
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
Description (incidence and severity):
The number of corpora lutea, implantations, total litter size, pre-implantation loss and pre-birth loss did not differ significantly between groups. Gestation length was also comparable between groups. No differences were observed in the pre-coital interval, copulatory and fertility indices between control and treated groups.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects obseved in the study
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed in the study
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical signs of pups such as pallor, cold to touch, small and/or bruise muzzle, were observed in control, mid- and high dose groups. No toxicological relevance was attributed to these signs since they were seen in treated as well as in control groups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
All pregnant females gave birth to live pups with the exception of one high dose female which had a total litter loss on day 3 post-partum. This female and two control females had unilateral implantation but was not treatment related.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Litter data including mean litter and pup weights were comparable between groups. Sex ratio of pups showed a slight increased number of males in high dose group respect to control. No toxicological relevance was attribute to the statistical significant increase observed on Day 4.
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:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Decedent pups were generally autolysed. No signs were seen in pups sacrificed on Day 4 post partum.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
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 adverse effects observed in the study
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Table 1: Fate of females: Group incidence

 

Treatment (mg/kg/bw/d)

0

100

300

1000

Initial group site

10

10

10

10

Unilateral Implantation

2

0

0

1

Total litter loss

0

0

0

1

With live pups on day 4 post-partum

10

10

10

9

Table 2: Implantation, pre-implantation loss data, pre-birth loss data and gestation length of females – Group mean data

Treatment (mg/kg/bw/d)

 

Corpora Lutea

Implantations

Total Litter size at birth

Pre-implantation loss %

Pre-birth loss %

Gestation length (days)

0

Mean

18.40

18.10

15.60

1.44

12.70

22.10

 

SD

3.37

3.18

5.27

3.17

25.82

0.32

 

n

10

10

10

10

10

10

 

100

Mean

16.30

15.80

14.20

3.26

10.09

22.10

 

SD

3.23

3.61

3.49

8.44

8.48

0.32

 

n

10

10

10

10

10

10

 

300

Mean

16.90

16.90

15.60

0.00

7.65

22.0

 

SD

1.97

1.97

2.17

0.00

7.35

0.00

 

n

10

10

10

10

10

10

 

1000

Mean

18.10

17.40

16.40

6.16

8.21

22.10

 

SD

5.17

5.32

5.44

12.73

9.93

0.32

 

n

10

10

10

10

10

10

Table 3: Litter data at birth, on day 1 and on day 4 post-partum of pregnant females – Group mean data

Treatment (mg/kg/bw/d)

 

At birth

On day 1 post-partum

On day 4 post-partum

Total litter size

Live litter size

Pup loss (%)

Litter weight (g)

Mean pup weight (g)

Live litter size

Cumulative loss (%)

Litter weight (g)

Mean pup weight (g)

0

Mean

15.60

15.40

1.13

101.62

7.00

14.40

6.37

132.38

9.61

SD

5.27

5.19

2.40

30.0

1.09

4.60

7.58

36.25

1.58

n

10

10

10

10

10

10

10

10

10

100

Mean

14.20

14.20

0.00

96.87

7.01

14.10

0.56

145.70

10.51

SD

3.49

3.49

0.00

19.08

0.81

3.38

1.77

29.27

1.08

n

10

10

10

10

10

10

10

10

10

300

Mean

15.60

15.60

0.00

106.70

6.94

14.80

5.51

143.13

9.76

SD

2.17

2.17

0.00

13.11

0.44

2.66

5.60

22.54

0.97

n

10

10

10

10

10

10

10

10

10

1000

Mean

16.40

16.20

5.50

115.50

7.10

15.60

13.30

163.63

9.46

SD

5.44

5.67

15.71

41.59

0.55

5.82

30.67

22.38

0.74

n

10

10

10

10

10

10

10

9

9

Table 4: Sex ratio of pups – Group mean data

Treatment (mg/kg/bw/d)

 

At birth

On day 4 post-partum

Males

Females

Total

% Males

Males

Females

Total

% Males

0

Mean

6.90

8.70

15.60

49.67

6.40

8.00

14.40

49.66

SD

2.02

4.03

5.27

19.96

1.71

3.59

4.60

19.93

n

10

10

10

10

10

10

10

10

100

Mean

6.90

7.30

14.20

50.50

6.80

7.30

14.10

50.14

SD

1.79

2.95

3.49

15.38

1.81

2.95

3.38

15.72

n

10

10

10

10

10

10

10

10

300

Mean

6.20

9.40

15.60

39.23

5.90

8.90

14.80

39.36

SD

2.20

1.65

2.17

11.28

2.08

1.66

2.66

10.29

n

10

10

10

10

10

10

10

10

1000

Mean

9.10

7.30

16.40

55.58

9.56*

7.78

17.33

55.66

SD

3.57

3.62

5.44

12.47

2.24

2.86

2.06

12.97

n

10

10

10

10

9

9

9

9

*= mean value of group is significantly different from control 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 1) and consitent studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on common precursors and breakdown products of hydrolysis and consistent trends in environmental fate, ecotoxicological and toxicological profile (refer to endpoint discussion for further details). The selected study is sufficient to fulfil the standard information requirements set out in Annex VIII, 8.5, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Analogue justification

No data on the reproduction toxicity of Fatty acids, C14-22, C16-24-alkyl esters (CAS 92797-30-3) are available. The assessment was therefore based on studies conducted with analogue substances as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the read across approach is provided in the technical dossier (see IUCLID Section 13).

 

Toxicity to reproduction

CAS 17671-27-1

A combined repeated dose toxicity and reproduction/developmental toxicity screening study (according to OECD 422) was performed using docosyl docosanoate (Key, 2014). The test item was administered orally (gavage) once daily at the doses of 100, 300 and 1000 mg/kg bw/day to male and female rats from two weeks before pairing to day 4 postpartum for females and to 5 weeks post-coitum for males.

No treatment-related parental effects were seen on viability, clinical signs, body weight (gain), food consumption, clinical chemistry parameters, during observational screening, and during macroscopic and microscopic examinations. In females, an increase in bilirubin, cholesterol and triglyceride levels was recorded at 300 and 1000 mg/kg bw/day. As this was only observed in one sex and no other hepatic changes were observed, this is not considered to be toxicologically relevant. Lower locomotor activity was recorded in males at 100 mg/kg and in females at 300 and 1000 mg/kg bw/day. As no effects were noted on other neurological parameters, this is not considered to be toxicologically relevant. Therefore the NOAEL for parental systemic toxicity was ≥ 1000 mg/kg bw/day.

In parental animals, no effects on reproductive function (qualitative sperm staging, oestrus cycle) or performance (male and female mating and fertility indices, conception index, precoital interval, and number of corpora lutea and implantation sites, gestation length) were observed, compared with the control animals. One female from the control group had a 100% postimplantation loss, which is considered to be incidental. The testis weight, epididymis weight, and histological examination of the testes in males as well as the weight and histological examination of the uterus and ovaries in females did not reveal any substance-related effects in the parental animals. Therefore, a NOAEL for parental fertility of ≥ 1000 mg/kg bw/day was derived for male and female rats.

CAS 22393-85-7

A combined repeated dose toxicity study and reproduction/developmental toxicity screening test, performed according to OECD 422 and GLP, is available (Supp., 2014). 10 rats/sex/dose were administered 100, 300 and 1000 mg/kg bw/day of tetradecyl oleate, formulated in carboxymethylcellulose (0.5% in purified water), once daily for at least 28 to 29 days (males) and up to 54 days (females) via gavage. The application started two weeks before mating on test Day 1 and ended on the day of, or one day before sacrifice. Day of sacrifice was on test Day 28 to 29 for the male rats and on Day 4 post-partum for the female rats, except for one single female, which was killed the day after the occurrence of total litter loss. In the main study 3 rats/sex/dose were administered 0, 100, 300 and 1000 mg/kg bw/day tetradecyl oleate for 14 days.

No treatment-related parental effects were seen on viability, clinical signs, body weight (gain), food consumption, haematological parameters, clinical chemistry parameters, during observational and neurological screening, and during macroscopic and microscopic examinations. Therefore the NOAEL for parental systemic toxicity was ≥ 1000 mg/kg bw/day.

In parental animals, no effects on reproductive function (qualitative sperm staging, oestrus cycle) or performance (male and female mating and fertility indices, conception index, pre-coital interval, and number of corpora luteae and implantation sites, gestation length) were observed, compared with the control animals. The testis weight, epididymis weight, and histological examination of the testes in males as well as the weight and histological examination of the uterus and ovaries in females did not reveal any substance-related effects in the parental animals. All the pregnant females gave birth to live pups with the exception of one high dose female which had a total litter loss on day 3 post-partum. This female and two females in the control group had unilateral implantation, which is not considered to be treatment-related. Therefore, a NOAEL for parental fertility of ≥ 1000 mg/kg bw/day was derived for male and female rats. 

Overall conclusion for toxicity to reproduction

There are no available studies on the toxicity to reproduction of Fatty acids, C14-22, C16-24-alkyl esters. Therefore analogue read across from 2 source substances was applied. No effects on reproductive parameters/organs were observed in the available screening studies. NOAEL values for reproduction toxicity were all at or above the currently applied limit dose of 1000 mg/kg bw/day. Therefore, no hazard to reproduction was identified. Based on the available data and following the analogue approach, Fatty acids, C14-22, C16-24-alkyl esters is not expected to be toxic to reproduction/fertility.

 

Effects on developmental toxicity

Description of key information

In the absence of data on development on target substance Fatty acids, C14-22, C16-24-alkyl esters an analogue read-across approach was conducted on suitable source substances:

Oral: OECD 422, rat, NOAEL development ≥ 1000 mg/kg bw/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 1) and consistent studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on common precursors and breakdown products of hydrolysis and consistent trends in environmental fate, ecotoxicological and toxicological profile (refer to endpoint discussion for further details). The selected study is sufficient to fulfil the standard information requirements set out in Annex VIII, 8.5, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Analogue justification

No data on the developmental toxicity/teratogenicity of Fatty acids, C14-22, C16-24-alkyl esters (CAS 92797-30-3) are available. The assessment was therefore based on studies conducted with analogue substances as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the read across approach is provided in the technical dossier (see IUCLID Section 13).

Developmental toxicity/teratogenicity

CAS 17671-27-1

A combined repeated dose toxicity and reproduction/developmental toxicity screening study (according to OECD 422) with docosyl docosanoate is available (Key, 2014). The test item was administered orally (gavage) once daily at the doses of 100, 300 and 1000 mg/kg bw/day to male and female rats from two weeks before pairing to day 4 postpartum for females and to 5 weeks postcoitum for males.

No toxicologically relevant treatment related adverse effects were seen regarding viability, body weight, gross pathology, sex ratio, feeding, morphological examinations including behaviour observations of the F1 offspring. Postural reflexes were determined on Day 1 postpartum in the pups. A significantly lower percentage of foetuses with positive response in the surface-righting reflex (righting reflex) at 1000 mg/kg bw/day was recorded compared to the control group. There were no treatment-related differences compared with the control animals at 100 or 300 mg/kg bw/day. The clinical relevance of this parameter remained unclear. Righting reflex was only assessed on Day 1 postpartum. The study report did not report the exact time point after birth (hours) when the test was conducted, nor considered biologically possible range of gestation length. No follow-up behavioural testing at later time points during development of pups was done. Testing of righting reflex is not required following OECD GL 422. The results of this parameter were therefore disregarded. Therefore, a developmental NOAEL of > 1000 mg/kg bw/day was determined based on the absence of toxicologically relevant effects.

CAS 22393-85-7

A combined repeated dose toxicity and reproduction/developmental toxicity screening study, performed according to OECD 422 and under GLP conditions is available (2014). 10 rats/dose/day were administered 100, 300 and 1000 mg/kg bw/day of tetradecyl oleate, formulated in carboxymethylcellulose (0.5% in purified water), once daily for at least 28 to 29 days (males) and up to 54 days (females) via gavage. The application started two weeks before mating on test Day 1 and ended on the day of, or one day before sacrifice. Day of sacrifice was on test Day 28 to 29 for the male rats and on Day 4 post-partum for the female rats, except for one single female, which was killed the day after the occurrence of total litter loss.

No toxicologically relevant treatment-related adverse effects were seen on viability, clinical signs, body weight, sex ratio, and gross pathology of the F1 offspring. Based on these results, a developmental NOAEL of > 1000 mg/kg bw/day was determined. 

Overall conclusion for developmental toxicity/teratogenicity

There are no available studies on the developmental toxicity and teratogenicity of Fatty acids, C14-22, C16-24-alkyl esters. Therefore analogue read across from 2 source substances was applied from a combined repeated dose toxicity study with the reproduction / developmental toxicity screening test. No hazard to development was identified. NOAEL values for developmental toxicity were all at or above the currently applied limit dose of 1000 mg/kg bw/day. Based on the available data and following the analogue approach, Fatty acids, C14-22, C16-24-alkyl esters is not expected to be hazardous for in utero development.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Fatty acids, C14-22, C16-24-alkyl esters (CAS 92797-30-3), data will be generated from data available for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Based on the analogue read-across approach the available data on toxicity to reproduction does not meet the classification criteria according to Regulation (EC) 1272/2008. However, as no prenatal developmental toxicity study is available, the conclusion for classification is ‘data lacking’.

Additional information