Fatty acids, C8-10, C8-10-alkyl esters

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral: OECD 422, rat, NOAEL fertility ≥ 1000 mg/kg bw/day (read-across)
Oral: OECD 422, rat , NOAEL systemic ≥ 1000 mg/kg bw/day (read-across)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Jun - 04 Aug 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)

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

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.

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

Key result

Critical effects observed:

no

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.

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 

Conclusions:

The source substance Tetradecyl-Oleate (CAS 22393-85-7) was tested in a combined repeated dose toxicity and reproduction/developmental toxicity screening study. A NOAEL of ≥ 1000 mg/kg bw/day for parental systemic toxicity and also for parental fertility was derived for male and female rats under the described test conditions.

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 precursors and breakdown products of hydrolysis and consistent trends in environmental fate, ecotoxicological and toxicological profile (refer to the endpoint discussion for further details). The available data is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 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 on the reproduction toxicity of Fatty acids C8-10, C8-10 alkyl esters (CAS 129677-93-6). 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 grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Section 13) and within Chapter 5.1 of the CSR.

Toxicity to reproduction

CAS 22393-85-7

A combined repeated dose toxicity and reproduction/developmental toxicity screening study was performed according to OECD Guideline 422 under GLP conditions (Key, 2014). 10 rats/sex/dose were administered 0, 100, 300 and 1000 mg/kg bw/day Tetradecyl oleate (CAS 22393-85-7) once daily, via gavage. Males were exposed for 28 - 29 days, from two weeks before mating on test day one until after mating. Females were exposed for 54 days (during 2 weeks prior to mating, during mating, during post-coitum, and during 3 days of lactation).

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, precoital interval, and number of corpora lutea 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 effects on fertility

There are no available studies on the toxicity to reproduction and fertility of Fatty acids C8-10, C8-10 alkyl esters. Therefore analogue read-across from a source substance was applied.

The potential for reproductive toxicity of the source substance (Tetradecyl oleate, CAS 22393-85-7) was assessed in a reproductive/developmental screening study (OECD 422). The NOAEL value for fertility was at the currently applied limit dose value of 1000 mg/kg bw/day. Therefore, no hazard to reproduction was identified. Based on the available data and following the analogue approach, the target substance Fatty acids C8-10, C8-10 alkyl estersis considered to not affect fertility.

Regulation (EC) No. 1907/2006, Annex IX, 8.7.3 Column 1, states that an extended one-generation reproduction toxicity study (OECD 443, basic test design or with additional modules) is required in one species, using the most appropriate route of administration, and having regard to the likely route of human exposure, ‘if the available repeated dose toxicity studies (e.g. 28-day or 90-day studies. OECD 421 or 422 screening studies) indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity’.

Therefore, in accordance with Annex IX, 8.7.3, Column 1, the registrant has considered the need to perform an extended one-generation reproduction toxicity study. In conclusion, information is available from the physico-chemical characteristics of the source and target substances and from studies performed with source substances of varying chain length and branching patterns, under different guidelines connected to reproduction and fertility. It is reasonable to assume for the source substance Fatty acids C8-10, C8-10 alkyl esters (CAS 129677-93-6), that the lack of reproductive effects noted in the available studies will reflect the results of reproductive parameters assessed specifically in an extended one-generation reproduction toxicity study. Therefore, referring to Regulation (EC) No. 1907/2006, Annex IX, 8.7.3, Column 1, performing an extended one-generation reproduction toxicity study (basic test design or with additional modules) is not scientifically necessary and, considering concerns regarding the use of vertebrate animals for experimental purposes, unjustified (please see further details in the IUCLID Section 7.8.1 'data waiving').

Effects on developmental toxicity

Description of key information

Oral: OECD 414, rat, NOAEL development ≥ 1000 mg/kg bw/day (read-across)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 Jun - 21 Jul 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

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

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:

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).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

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.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Visceral malformations:

no effects observed

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.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: teratogenicity

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: embryotoxicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

exposure duration was only from day 6-15 of gestation instead of day 5-19

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

yes

Remarks:

Exposure duration was only from day 6-15 of gestation instead of day 5-19.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: approx. 8-10 weeks
- Weight at study initiation: mean approx. 197 g
- Housing: individually in Makrolon Type M3 cages (Ebeco) with standard softwood bedding (ARWI-Center, Essen, Germany)
- Diet: Pelleted Altromin Maintenance Diet 1324, Lot No. 221092/1558 (Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 40-56
- Air changes (per hr): 10-15 per hr
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: Arachidis oil, DAB 10

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing solutions were prepared daily before administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility of test substance
- Concentration in vehicle: 20, 60 and 200 mg/mL, respectively for the 100, 300 and 1000 mg/kg bw dose groups
- Amount of vehicle (if gavage): 5 mL/kg bw

After arrival all females were assigned to the different groups using a computer-generated random algorithm.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant, at day 0
- Proof of pregnancy: vaginal plug day 0 of pregnancy

Duration of treatment / exposure:

from day 6 up to day 15 of gestation

Frequency of treatment:

once daily

Duration of test:

until day 20 of gestation

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:

24 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Other:
group 1: 0 mg/kg bw/day
group 2: 100 mg/kg bw/day
group 3: 300 mg/kg bw/day
group 4: 1000 mg/kg bw/day

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
Mortality rate: The animals were checked at least twice daily for any mortality.
Signs and/or symptoms: The animals were observed at least twice daily (working days) for signs of reaction to treatment and/or symptoms of illness.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Post mortem examination, including gross macroscopic examination of all maternal organs, with emphasis on the uterus, uterine contents, position of fetuses in the uterus and number of corpora lutea, was performed and the data recorded.
The uteri (including content) of all females were weighed at necropsy on day 20 post coitum to enable the calculation of the corrected body weight gain.
- Any female sacrificed or found dead during the study was subjected to macroscopic examination of the visceral organs, with emphasis on the uterus and its content.

BODY WEIGHT: Yes, mean body weight changes
- Time schedule for examinations: days 0, 6, 16 and 20 of gestation

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

Number and distribution of intrauterine implantations were classified as live or death fetuses, late intrauterine deaths (resorptions), early intrauterine (resorption sites). The fetuses were removed from the uterus. Intrauterine deaths were classified on the basis of the presence (late) or absence (early) of fetal or decidual tissue in addition to placental tissue.

Fetal examinations:

- External examinations: Yes: half per litter
- Soft tissue examinations: Yes: half per litter: malformations oh hydrocephalus, variations of brain, adrenal gland, renal pelvis, ureter
- Skeletal examinations: Yes: half per litter: malformations of hydrops, retardations of skull bones, hyoid, sternebrae, pelvis, 13th rib
- Head examinations: Yes: half per litter

The live fetuses were sexed, weighed individually including placentae, examined for gross external abnormalities and allocated to one of the following procedures:
1) Half of the fetuses from each litter was non individually fixed in Bouin's solution in order to examine viscera and brain by Wilson's slicing technique. After examination the sections were not preserved.
2) The remaining fetuses were placed non individually in a solution of potassium hydroxide for clearing and were stained with alizarin red (Shandon Varistain 24-T). The skeletons were examined and preserved in plastic containers. All abnormalities were recorded.

Statistics:

The following statistical methods were used:
If the variables could be assumed to follow a normal distribution, the Dunnett-Test, based on a pooled variance, was applied for the comparison between the treated groups and the control group, otherwise the Steel-Test was applied.
Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information (Bonferroni-Holm-corrected).

Historical control data:

Findings both on the individual foetus and on the litter basis did not differ from the available historical control obtained in six developmental toxicity studies on the same species.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight profiles of the pregnant females were essentially similar in all groups. Mean corrected body weight gain of the treatment groups compared favourably with the control values.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
The dams tolerated the applied dose levels of up to 1000 mg/kg bw/day without lethality and clinical signs of systemic toxicity.
Maternal body weight gain was not affected by the treatment.

Mortality:
No death occurred in the dams of group 1 (vehicle control) and in the test groups 2 - 4.

Signs and/or symptoms:
No compound-related symptoms were observed in all treatment groups. In one female (group 3) was noted a skin incrustion on the back and another female (group 1) was severely aggressive by handling.

Body weight gains and corrected body weight:
Body weight profiles of the pregnant females were essentially similar in all groups. Mean corrected body weight gain of the treatment groups compared favourably with the control values.

Reproduction data:
No compound-related differences were noted between the mean reproduction data of the test groups in comparison to the control group. In the group 2 and 4 the post-implantation loss and total embryonic deaths were significantly decreased. These findings were considered to be incidental because of the high control values. Furthermore the number of total fetuses was increased in the group 2 and 4, which is also incidental because there was no dose-relationship.

Necropsy findings:
No macroscopic changes were noted in the dams of the groups 1 - 4.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Apart from dose group 1000 mg/kg bw/day (one dead foetus) all females had viable foetuses. Pre- and postimplantation loss and mean numbers of resorption were unaffected by treatment. All parameters were comparable with the animals of the control group. Skeletal and visceral investigations detected no treatment-related malformations.

Body weight:
The weights of live fetuses exibited no significant differences on a litter and individual basis e.g. mean weight between the control group and the treatment groups.

Placenta and uterus weight:
The weights of placentae and the whole uterus showed no significant differences between the control group and the treatment groups.

Sex ratios:
The sex ratio of the fetuses was not effected by the treatment with the test substance.

External examinations:
No substance-related macroscopical findings were noted at external examination of fetuses which were considered to be an effect of the treatment with the test article. In the group 1 was noted a beginning hydrops and in the group 4 one fetus with paleness and one dead fetus.

Visceral examination:
The findings were as follows:
Group 1: 127 examined fetuses
28 hydronephrosis
9 ureter dilatation
5 ureter waved
1 runt, brain lateral sinus dilatation, other organs normal
1 thorax - blood coagulum [artifact]
1 adrenal central pinhead cyst [suspicious]
1 umbilical region - gut protrusion [artifact]

Group 2: 138 examined fetuses
34 hydronephrosis
12 ureter dilatation
3 ureter waved
1 runt, hydrocephalus internus

Group 3: 138 examined fetuses
26 hydronephrosis
5 ureter dilatation
6 ureter waved
1 ear region subcutaneous hematoma
1 umbilical region - gut protrusion [artifact]

Group 4: 140 examined fetuses
24 hydronephrosis
10 ureter dilatation
8 ureter waved
1 inguinal hernia, protrusion of gut and testis between peritoneum and trunk, muscles [artifact]
1 runt, brain lateral sinus dilatation, other organs normal

The visceral examination of the preserved fetuses did not reveal any treatment-related abnormalities.

Skeletal examination of fetuses:
Retardations:
Group 1: 141 examined fetuses
Group 2: 152 examined fetuses
single sternebrae non ossified,
significant increase at level 1% (34 fetuses out of 22 dams)
Group 3: 150 examined fetuses: no significant findings
Group 4: 152 examined fetuses
single sternebrae non ossified,
significant increase at level 5 % (29 fetuses out of 22 dams)
two sternebrae non ossified,
significant increase at level 1 % (21 fetuses out of 22 dams)

The statistically significant differences were considered to be incidental because these retardation effects were not accompanied by weight retardation of the treatment groups. The incidental character of these retardations is emphasized by the fact the values were within the normal range of variation for this strain.

Variations (examined fetuses):
Group 1: no variations
Group 2: no variations
Group 3: no variations
Group 4: no variations

Malformations (examined fetuses):
Group 1: 1 fetus beginning hydrops
Group 2: no findings
Group 3: no findings
Group 4: no findings

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: embryotoxicity

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: teratogenicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Fatty acids, C16-18, 2-ethylhexyl esters up to a dose of 1000 mg/kg bw/day does not produce any embryo- and foetotoxic or teratogenic effects. The NOAEL for maternal-, developmental-, embryo-, foetotoxicity and teratogenicity is deduced ≥ 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises two adequate and reliable studies (Klimisch score 2) from reference substances with similar structures 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 the endpoint discussion for further details). The selected studies are thus sufficient to fulfil the standard information requirements set out in Annex VIII-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

Data on developmental toxicity of Fatty acids C8-10, C8-10 alkyl esters are not 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 analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

 

Developmental toxicity/teratogenicity

 

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 study performed using a study protocol similar to OECD guideline 414 (WoE, 1997). 23-24 pregnant female Sprague-Dawley rats/dose level were administered 0, 100, 300 and 1000 mg/kg bw/day of the test substance by gavage on gestation Day 6 to 15.

No signs of systemic toxicity were observed in the P-females and no treatment-related effects were observed. 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 mid-dose group (300 mg/kg bw/day). 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 developmental is considered to be ≥ 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. Based on the available data it is not considered necessary to perform an additional, dedicated, developmental toxicity study.

 

CAS 91031-48-0

A developmental toxicity study was performed according to OECD Guideline 414 under GLP conditions (WoE, 1994). Twenty-four female Sprague-Dawley rats per dose were administered 0, 100, 300 and 1000 mg/kg bw/day Fatty acids, C16-18, 2-ethylhexyl esters (CAS 91031-48-0) once daily from day 6 up to day 15 of gestation via gavage in arachidis oil as vehicle. Maternal animals were examined for clinical signs, mortality, body weight changes, and gross pathology. Number of abortions, pre- and post-implantation losses, total litter losses, duration of pregnancy, early and late resorptions, dead foetuses, pregnancy rate, placenta and uterus weight were assessed. In foetuses body weight changes, number of live offspring, sex ratio, external, visceral and skeletal malformations were assessed.

No adverse effects on maternal toxicity, maternal developmental toxicity and foetuses were observable up to the limit dose of 1000 mg/kg/day.

Based on the absence of adverse toxic effects, the NOAEL for developmental toxicity/teratogenicity and for maternal developmental toxicity is considered to be 1000 mg/kg bw/day.

 

CAS 22393-85-7 

A combined repeated dose toxicity and reproduction/developmental toxicity screening study was performed according to OECD Guideline 422 under GLP conditions (Rossiello, 2014). 10 rats/ sex/dose were administered 0, 100, 300 and 1000 mg/kg bw/day Tetradecyl oleate (CAS 22393-85-7) once daily, via gavage. Males were exposed for 28-29 days, from two weeks before mating on test day one until after mating. Females were exposed for 54 days (during 2 weeks prior to mating, during mating, during post-coitum, and during 3 days of lactation).

The results of the offspring (viability) parameters pre-implantation loss, pre-birth loss, stillbirths, live births, cumulative pup loss on day 4 post-partum, litter size and pup weight were comparable between control and treatment group. Clinical signs of pups such as pallor, cold to touch, small and/or bruised muzzle, were observed in control and treatment groups and were therefore not considered to be of toxicological relevance. The number of male pups was significantly increased compared with the control group on Day 4 only. As the sex ratio (absolute percentage of males to females) was not significantly different from control, this is not considered to be a toxicologically relevant effect. The gross pathological examinations did not reveal any treatment-related effects. Based on the lack of effects, the NOAEL for developmental toxicity/teratogenicity is considered to be ≥ 1000 mg/kg bw/day.

 

Overall conclusion for developmental toxicity/teratogenicity

The potential for developmental toxicity and teratogenicity of the source substance (Tetradecyl oleate, CAS 22393-85-7) was assessed in a reproductive/developmental screening study (OECD 422).  Further, isononanoic acid, C16-18 alkyl esters (CAS 111937-03-2) and Fatty acids, C16-18, 2-ethylhexyl esters (CAS 91031-48-0) were tested for developmental toxicity according to OECD 414. In all studies, NOAEL values for developmental toxicity/teratogenicity were at the currently applied limit dose value of 1000 mg/kg bw/day. Therefore, no hazard to reproduction was identified. Based on the available data and following the analogue approach, the target substance Fatty acids C8 -10, C8 -10 alkyl esters is considered to be not toxic to 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 C8-10, C8-10 alkyl esters (CAS 129677-93-6), data will be generated from data 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, and is therefore conclusive but not sufficient for classification.

An OECD Guideline 422 study will not provide evidence for definite claims of no reproduction/developmental effects. However, since no adverse effects on reproduction and development was noted up to and including the limit dose of 1000 mg/kg bw/day in all available studies (including the two developmental toxicity studies), the conclusion of no classification (conclusive but not sufficient for classification) is considered to be justified.