Fatty acids, C14-18, C14-18-alkyl esters

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

06 Jun - 04 Aug 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP-guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2012)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

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

Remarks:

Doses / Concentrations:
100, 300 1000 mg/kg bw/day
Basis:
actual ingested

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

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

No relevant clinical signs or mortality were observed in males and females throughout the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

No difference of toxicological significance were seen in body weight or body weight gain. No intergroup differences were seen in food consumption.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)

No relevant difference in oestrous cycle was observed in treated females when compared to controls.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)

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 (PARENTAL ANIMALS)

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.

ORGAN WEIGHTS (PARENTAL ANIMALS)

No relevant differences in terminal body weight or organ weights were seen between the controls and treated animals of both sexes

GROSS PATHOLOGY (PARENTAL ANIMALS)

No remarkable changes were noted at post mortem examination in treated animals when compared with controls.

HISTOPATHOLOGY (PARENTAL ANIMALS)

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.

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

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

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

VIABILITY (OFFSPRING)

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. This finding was considered incidental since it was observed also in two control females.

CLINICAL SIGNS (OFFSPRING)

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.

BODY WEIGHT (OFFSPRING)

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.

GROSS PATHOLOGY (OFFSPRING)

Decedent pups were generally autolysed. No signs were seen in pups sacrificed on Day 4 post partum.

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

Reproductive effects observed:

not specified

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

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 2 due to read-across) from a reference substance with similar structure and intrinsic properties. Read-across is justified based on common functional groups; common precursors/breakdown products and similarities in physicochemical and toxicological properties (refer to endpoint discussion for further details). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, 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

Justification for read-across

There are no data available on the toxicity to reproduction of fatty acids, C14-18, C14-18-alkyl esters (CAS 85566-24-1). In order to fulfil the standard information requirements set out in Annex IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from structurally related substances was conducted.

In accordance with Article 13 (1) of Regulation EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across to avoid the need to test every substance for every endpoint).

The target and source substances are considered to be similar on the basis of the structural similar properties and/or activities. The available endpoint information on the source substance is used to predict comparable results for the target substance fatty acids, C14-18, C14-18-alkyl esters (CAS 85566-24-1).

The target substance is characterized as an UVCB substance comprised of esters of mainly C14 fatty acid/C14 alcohol, C16 fatty acid/C14 alcohol and C16 fatty acid/C16 alcohol substances. The source substance is structurally similar to the target substance: Tetradecyl oleate (CAS 22393-85-7) is a UVCB substance consisting of C18:1 oleic acids esterified with C14 alcohol.  Thus, target and source substance contain similar structural properties based on common functional groups.

A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

 

 

CAS 22393-85-7

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, according to OECD 422 and GLP is available (Rossiello, 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 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 parental animals no test item-related signs of toxicity were observed during the observational and neurological screenings. No differences of toxicological significance were seen in terminal body weight, body weight gain and in food consumption. No relevant changes were recorded in parental animals on clinical pathology investigations (haematology and clinical chemistry). Macroscopic inspection at autopsy and histopathological examination revealed no test item related changes.

The mating performance including the pre-coital interval and the copulatory evidence did not show any differences between groups. The resulting copulatory and fertility indices were 100% in all groups. All females were pregnant and had comparable length of gestation. No variations were found in the number of corproa lutea and implantations between groups. 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. This finding was considered incidental since it was observed also in two control females. The litter size at birth and on Day 4 post-partum was also similar between groups as well as the mean litter and pup weights. Statistically significant increased mean group sex ratio for males was seen in high dose group on Day 4. No relevant findings in pups were found at daily clinical observation or at post-partum examination. On the basis of the results obtained in the study, the NOAEL for systemic toxicity and reproduction/developmental toxicity was considered to be ≥1000 mg/kg bw/day for males and females.

Conclusions for reproductive toxicity 

Based on read-across data, sufficient evidence is available to conclude that the substance fatty acids, C14-18, C14-18-alkyl esters (CAS 85566-24-1) has no toxic effects to reproduction.

 

Short description of key information:
Oral (OECD 422), rat: NOAEL (fertility) ≥ 1000 mg/kg bw/d
Oral (OECD 422), rat: NOAEL (systemic) ≥ 1000 mg/kg bw/d

Justification for selection of Effect on fertility via oral route:
Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is an adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

Effects on developmental toxicity

Description of key information

Oral (OECD 414), rat, oral:
NOAEL (F1) ≥ 1000 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

28 Jun - 21 Jul 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study with acceptable restrictions. Short treatment period (Day 6-15 of gestation), body weight was recorded on Day 0, 6, 16 and 20 only, food consumption was not recorded, the analytical purity of the test substance was not specified.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

exposure from Day 6 to Day 15 of gestation, body weight was recorded on Day 0, 6, 16 and 20, food consumption was not recorded

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Sprague-Dawley, CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: 202.6 ± 20.4 g - 219 ± 25.6 g (range of group mean values)
- Housing: the animals were housed individually in Makrolon Type M3 cages (Ebeco, Castrop-Rauxel, Germany), on standard softwood bedding (ARWI-Center, Essen, Germany)
- Diet: pelleted Altromin Maintenance Diet 1324 (Fa. Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 48-82
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12 (lux units 50 - 550)

IN-LIFE DATES: From: 28 Jun 1994 To: 21 Jul 1994

Route of administration:

oral: gavage

Vehicle:

other: 0.5% sodium carboxymethylcellulose + 0.25% Cremophor in aqua dest.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared daily before administration, adjusted to the body weight measured on Day 6 of gestation.

VEHICLE
- Concentration in vehicle: 1, 3, 10% (10, 30, 100 mg/mL)
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mixture of the test item was analysed once to verify the actual concentration. The measured concentration was within the expected range. The nominal concentrations 1 % (100 mg/kg bw/day), 3 % (300 mg/kg bw/day) and 10 % (1000 mg/kg bw/day) were measured to be 1.1%, 3.0% and 10.5%, respectively.

Details on mating procedure:

- Any other deviations from standard protocol: Primiparous time-mated females were used. The females were mated at the supplier with an accurate day of mating and received at the testing facility on gestation day 0.

Duration of treatment / exposure:

Day 6-15 of gestation

Frequency of treatment:

daily, 7 days/week

Duration of test:

10 days; Day 6-15 of gestation

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
other: nominal dose

No. of animals per sex per dose:

23 P females (100 and 300 mg/kg bw/day)
24 P females (control, 1000 mg/kg bw/day)

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 0 (prior to administration), 6, 16 and 20 of gestation

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: gross macroscopic examination of all reproductive and gender-specific organs, with emphasis on the uterus, uterine contents, position of the fetuses in the uterus and number of corpora lutea

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: weight of fetuses

Fetal examinations:

- External examinations: Yes, all fetuses
- Soft tissue examinations: Yes, half per litter (146 fetuses of group 1, 133 fetuses in group 2, 169 fetuses in group 3 and 162 fetuses in group 4)
- Skeletal examinations: Yes, half per litter (159 fetuses of group 1, 144 fetuses in group 2, 178 fetuses in group 3 and 172 fetuses in group 4)
- Head examinations: No

Group 1: control
Group 2: 100 mg/kg bw/day
Group 3: 300 mg/kg bw/day
Group 4: 1000 mg/kg bw/day

Statistics:

If the variables could be assumed to follow a normal distribution, the Dunnett-Test, based on pooled variance, was applied for comparion between groups. The Steel-Test was applied when the data could not be assumed to follow a normal distribution. Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information (Bonferroni-Holm-corrected).

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
There was no mortality during the study period. No signs of systemic toxicity were observed. The mean body weight of the mid-dose group was statistically significantly increased on Day 16 and 20 of the study period (see Table 1). This considered to be an incidental observation, as the body weight gain over the study period was comparable between the control and treatment groups (58.9, 67.8, 57.4 and 59.6 g for the control, 100, 300 and 1000 mg/kg bw/day group, respectively). One female in each of the control group and the low-dose group was not pregnant, while all the pregnant females had viable fetuses (see Table 2).

No substance-related effects on the reproductive parameters (number of corpora lutea, implantation sites, pre-implantation loss, post-implantation loss, embryonic deaths, embryonic resorptions, fetal resorptions, live fetuses, dead fetuses) were observed (see Table 3). The necropsy and macroscopic examination did not show any treatment-related effects. One female in the high-dose group had blood in the uterine horn, but this is not considered to be treatment-related as no other effects were observed.

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The external examination of the foetuses did not reveal any treatment-related macroscopical effects. The skeletal examination showed a statistically significant increase in the number of foetuses with 6 ossified sternebrae in the high-dose group (see Table 5). As there were no increases in ossification anywhere else and no overall increase in abnormal findings for this group, the result is considered to be incidental. The results for the remaining offspring parameters (body weight, placental weight, sex ratio) were comparable between the control and treatment groups.

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Body weights

Dose group	Control	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Gestation day 0	212.3	202.6	219.7	209.0
Gestation day 6	263.1	255.5	273.5	259.4
Gestation day 16	340.7	338.6	361.7*	341.5
Gestation day 20	403.3	400.7	428.0*	407.9

*Dunnett-Test based on pooled variance, p < 0.05

 

Table 2: Summary of mating performance of the females

Dose group	Control	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
No. of mated females	24	23	23	24
No. of pregnant females*	23	22	23	24
No. of premature litters**	0	0	0	0
No. of mortalities	0	0	0	0

*Included in the statistical analysis

**Premature litter is an event in the cage immediately before the caesarean section

 

Table 3: Reproduction parameters for dams with live foetuses

Dose group	Control	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Number of dams	23	22	23	24
Corpora lutea (total)	376	343	403	390
Corpora lutea (mean±SD)	16.3±1.5	15.6±1.2	17.5±1.6	16.2±2.3
Implantation sites (total)	316	301	367	346
Implantation sites^aas: -% of corp. lutea - mean±SD	84.0 13.7±3.7	87.8 13.7±2.5	91.1 16.0±1.9	88.7 14.4±3.1
Pre-implantation loss (total)^b	60	42	36*	44
Pre-Implantation lossas % of corpora lutea	16.0	12.2	8.9	11.3
Post-implantation loss^b	11	24	20	12
Post-implantation loss as % of implantation sites	3.5	8.0	5.4	3.5
Embryonic deaths total^b	11	24	20	12
Embryonic resorptions (total)^a	9	18	20	10
Embryonic resorptions as % of implantation sites (mean±SD)	2.8±0.4	6.0±0.8	5.4±0.9	2.9±0.4
Foetal resorptions (total)^a	2	6	0	2
Foetal resorptionsas % of implantation sites(mean±SD)	0.6±0.1	2.0±0.3	0	0.6±0.1
Fetuses per dam (mean±SD)	13.3±3.6	12.6±3.1	15.1±2.4	13.9±2.7
Live foetuses^a	305	277	347	334
Dead foetuses^a	0	0	0	0
Malformed foetuses	0	0	0	0
Uterus weight^c(mean±SD)	81.3±24.1	77.4±18.0	97.0±15.7*	88.8±18.3

^aSteel Test

^bFishers Exact Test (Bonferroni-Holm-Corrected)

^cDunnett-Test based on pooled variance

*p < 0.05

 

Table 4: Developmental parameters for offspring

Dose group	Control	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Number of live foetuses (m/f)	305 (154/151)	277 (141/136)	347 (162/185)	334 (165/169)
Sex ratio (m/f)	0.51/0.49	0.51/0.49	0.47/0.53	0.49/0.51
Weights of live foetuses (mean±SD)	4.1±0.8	4.1±0.6	4.4±0.8	4.2±0.6
No. of runts	1	2	1	2

Table 5: results of skeletal examination of offspring

Dose group	Control	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
No abnormal findings, number (% of total)	10 (6.3%)	12 (8.3%)	32 (18.0%)**	10 (5.8%)
6 ossified sternebrae, number (% of total)	124 (78.0%)	120 (83.3%)	151 (84.8%)	153 (89%)**

** Fishers Exact Test (two-sided), p < 0.01

 

Conclusions:

The test substance had no effect on intrauterine development
CLP: not classified
DSD: not classified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 2) from a reference substance with similar structure and intrinsic properties. Read-across is justified based on common functional groups; common precursors/breakdown products and similarities in physicochemical and toxicological properties (refer to endpoint discussion for further details). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7, 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

Justification for read-across

There are no data available on developmental toxicity of fatty acids, C14-18, C14-18-alkyl esters (CAS 85566-24-1). In order to fulfil the standard information requirements set out in Annex IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from structurally related substances was conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across to avoid the need to test every substance for every endpoint).

The target substance and all source substances are considered to be similar on the basis of the structural similar properties and/or activities. The available endpoint information on the source substances is used to predict comparable results for the target substance fatty acids, C14-18, C14-18-alkyl esters (CAS 85566-24-1).

The target substance is characterized as an UVCB substance comprised of esters of mainly C14 fatty acid/C14 alcohol, C16 fatty acid/C14 alcohol and C16 fatty acid/C16 alcohol substances. The two source substances are structurally similar to the target substance: Tetradecyl oleate (CAS 22393-85-7) is a UVCB substance consisting of C18:1 oleic acids esterified with C14 alcohol. Isononanoic acid, C16-18 alkyl esters (CAS 111937-03-2) is a UVCB substance consisting of isononanoic acid esterified with C16-18 alcohol. Thus, target and source substance contain similar structural properties based on common functional groups.

A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

 

CAS 111937-03-2

The potential of isononanoic acid, C16-18 alkyl esters (CAS 111937-03-2) to cause developmental toxicity was assessed in a prenatal developmental toxicity study (similar to OECD guideline 414) (Pitterman, 1997). 23-24 pregnant female Sprague-Dawley rats/dose level were administered 100, 300 and 1000 mg/kg bw/day of the test substance by gavage on gestation Days 6 to 15. On Day 20 the females were euthanized and examined.

No treatment-related systemic effects were observed in the P-females. One female in the high-dose group had blood in the uterine horn, but this is not considered to be treatment-related as no related effects were observed. One female in each of the control group and the low-dose group did not become pregnant, while all the pregnant females had viable foetuses. No treatment-related effects on the reproductive parameters (number of corpora lutea, implantation sites, pre-implantation loss, post-implantation loss, embryonic deaths, embryonic resorptions, foetal resorptions, live foetuses, dead foetuses) were observed. The external examination of the F1-foetuses did not reveal any treatment-related macroscopic findings. The skeletal examination showed a statistically significant increase in the number of foetuses with 6 ossified sternebrae in the high-dose group. As there were no increases in ossification anywhere else and no overall increase in abnormal findings for this group, the result is considered to be incidental. The results for the remaining offspring parameters (body weight, placental weight, sex ratio) were comparable between the control and treatment groups. The NOAEL for developmental toxicity and teratogenicity was set at ≥ 1000 mg/kg bw/day. The most recent version of OECD guideline 414, adopted in January 2001, stipulates that the dams should be dosed from implantation (gestation Day 0) to the day prior to scheduled caesarean section (around gestation day 19 for rats). The study was performed according to the previous version of OECD guideline 414, adopted in May 1981, which recommended exposure during organogenesis; gestation Day 6-15 for rats. The available data on repeated dose toxicity and developmental toxicity is of high quality and does not show treatment-related adverse effects at dose levels up to and including the highest dose of 1000 mg/kg bw/day, indicating that adverse effects are unlikely to occur in the period prior to Day 6 of gestation (Pitterman, 1993, 1997). Based on the available it is not considered necessary to perform an additional, dedicated, developmental toxicity study.

CAS 22393-85-7

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, according to OECD 422 and GLP is available (Rossiello, 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 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 parental animals, no test item-related signs of toxicity were observed during the observational and neurological screenings. No differences of toxicological significance were seen in terminal body weight, body weight gain and in food consumption. No relevant changes were recorded in parental animals on clinical pathology investigations (haematology and clinical chemistry). Macroscopic inspection at autopsy and histopathological examination revealed no test item related changes.

The mating performance including the pre-coital interval and the copulatory evidence did not show any differences between groups. The resulting copulatory and fertility indices were 100% in all groups. All females were pregnant and had comparable length of gestation. No variations were found in the number of corproa lutea and implantations between groups. 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.This finding was considered incidental since it was observed also in two control females. The litter size at birth and on Day 4 post-partum was also similar between groups as well as the mean litter and pup weights. Statistically significant increased mean group sex ratio for males was seen in high dose group on Day 4. No relevant findings in pups were found at daily clinical observation or at post-partum examination. On the basis of the results obtained in the study, the NOAEL for systemic toxicity and reproduction/developmental toxicity was considered to be ≥1000 mg/kg bw/day for males and females.

Conclusions for developmental toxicity 

Based on read-across data, sufficient evidence is available to conclude that the substance fatty acids, C14-18, C14-18-alkyl esters (CAS 85566-24-1) has no toxic effects on intrauterine development.

 

Justification for selection of Effect on developmental toxicity: via oral route:
Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is an adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment (refer to the endpoint discussion for further details).

Justification for classification or non-classification

Based on read-across from structurally similar substances, the available data on toxicity to reproduction do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.