Dodecyl nonan-1-oate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

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

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

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 2013 - 20 Oct 2014

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 1996

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted in 2016

Deviations:

yes

Remarks:

no endocrine disruptor endpoints adressed

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: 191 to 204 g (males) and 172 to 179 g (females)
- Housing: From arrival to pairing: animals were housed 5 of one sex to a cage, in polysulphone solid bottomed cages measuring 59.5x38x20 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating: animals were housed one male to one female in clear polysulphone cages measuring approximately 43x27x18 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy). Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating; the females were transferred to individual solid bottomed cages (Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy) for the gestation period and parturition.
- Diet: laboratory rodent diet, 4 RF 21 (Mucedola S.r.l., Settimo Milanese (MI), Italy), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 2 weeks

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

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous carboxymethylcellulose (0.5% CMC)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was suspended in the vehicle. Formulations were prepared daily.

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL for dose levels of 100, 300 and 1000 mg/kg bw/day, respectively
- Amount of vehicle: 10 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1 male to 1 female (monogamous).
- Length of cohabitation: The female was placed with the same male until pregnancy had occurred or 2 weeks had elapsed.
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- After 2 weeks of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged singly.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability of the test substance in the vehicle were verified by gas chromatopgraphy with a flame ionisation detection (FID). Concentration verification was conducted on a weekly basis.

Duration of treatment / exposure:

Males: The daily administration of the test item was started two weeks before mating and lasted until test day 28 to 29, which was one day before sacrifice.
Females: The daily administration of the test item was started two weeks before mating and continued until day 3 post-partum.
Maximum: 54 days of treatment.

Frequency of treatment:

once daily; 7 days/week

Details on study schedule:

not applicable for OECD 422 study

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on a 14-day range-finding study (Rossiello, 2013. RTC Study No.: 93730EXT)

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily during the study, each animal was observed and any clinical signs recorded.


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

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

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

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

OTHER:
Reproduction paramters: number of pregnant females, pre-coital time, gestation length

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily in the morning starting two weeks before pairing until a positive identification of copulation was made. The vaginal smear data were examined to determine the following: anomalies of the oestrous cycle and pre-coital interval (i.e., the number of nights paired prior to the detection of mating).

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight, and qualitative sperm staging.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed. A detailed qualitative evaluation of testes was performed on 5 randomly selected control and high dose males. The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: litter weight, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [males were sacrificed on day 29 or 30]
- Maternal animals: All surviving animals [females were sacrifices on day 4 post-partum or shorty thereafter]

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

HISTOPATHOLOGY: Yes, all organs that were included for fixation (5/sex of control and high dose group)

Postmortem examinations (offspring):

SACRIFICE
- The surviving F1 offspring were sacrificed at 4 days of age.


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

HISTOPATHOLOGY / ORGAN WEIGTHS
not performed

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test.
The criterion for statistical significance was p<0.05

Reproductive indices:

Male Copulatory Index (%) = No. of animals mated/No. of animals paired x 100
Male Fertility Index (%) = No. of males which induced pregnancy/ No. of males paired x 100
Female Copulatory Index (%) = No. of animals mated/No. of animals paired x 100
Female Fertility Index (%) = No. of pregnant females/No. of females paired x 100
Males and females:
Precoital interval = Mean number of days between pairing and mating

Offspring viability indices:

Pre-implantation loss [%] = (No. of corpora lutea - No. of implantations/ No. of corpora lutea) x 100
Pre-birth loss [%] = (No. of visible implantations - total litter size at birth/ No. of visible implantations
) x 100
Pup loss at birth [%] = (Total litter size - live litter size/ Total litter size) x 100
Cumulative pup loss on Day 4 post-partum [%] = (Total litter size at birth - live litter size at Day 4/ Total litter size at birth) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

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

Mortality:

no mortality observed

Description (incidence):

No mortality were observed in males and females throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No difference of toxicological significance were seen in body weight or body weight gain. No relevant differences in terminal body weight were seen between the controls and treated animals of both sexes.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No intergroup differences were seen in food consumption.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No changes of toxicological significance were observed. The statistically significant differences between control and treated animals (erythrocytes, mean corpuscular haemoglobin and leucocytes in males, haemoglobin, haematocrit and platelets in females) were of low magnitude and/or not dose-related, therefore considered incidental.
No changes were recorded at the coagulation tests.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant fluctuations of some biochemical parameters (mean group values) were recorded such as: increase of glucose in males dosed with 100 and 1000 mg/kg/day (48% for both dosages), increase in urea in those receiving 100 mg/kg/day (19%), increase of aspartate aminotransferases in females dosed with 300 mg/kg/day (35%), decrease of bilirubin (81%) and increase of potassium (10%) in those treated with 1000 mg/kg/day when compared to controls. Due to the lack of dose- and sex-consistency and to the absence of other relevant findings, the above alterations were considered unrelated to treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.
No significant differences were observed between groups, in motor activity, grip strength and sensory reactivity to stimuli, evaluated at the end of treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed. The lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under the experimental conditions.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed. The lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under the experimental conditions.

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

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

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Reproductive performance:

no effects observed

Description (incidence and severity):

The number of corpora lutea, implantations, total litter size, pre-implantation loss and pre-birth loss did not differ significantly between groups. Gestation length was also comparable between groups. No differences were observed in the pre-coital interval, copulatory and fertility indices between control and treated groups.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed in the study

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed in the study

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Similar signs such as pallor, cold to touch and/or bruise muzzle were recorded in pups of both control and high dose groups. Small foetuses were seen in mid- and high dose groups as well as in control. No toxicological relevance was attributed to the signs since they were present in treated as well as in control animals.

Mortality / viability:

mortality observed, non-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. This finding was considered incidental since it was observed also in two control females.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Litter data including mean litter and pup weights were comparable between groups.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

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

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Sex ratio of pups showed a slight increased number of males in high dose group. No toxicological relevance was attributed to this observation.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed in the study

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1: Fate of females: Group incidence

	Treatment (mg/kg/bw/d)
	0	100	300	1000
Initial group site	10	10	10	10
Unilateral Implantation	2	0	0	1
Total litter loss	0	0	0	1
With live pups on day 4 post-partum	10	10	10	9

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

Treatment (mg/kg/bw/d)		Corpora Lutea	Implantations	Total Litter size at birth	Pre-implantation loss %	Pre-birth loss %	Gestation length (days)
0	Mean	18.40	18.10	15.60	1.44	12.70	22.10
	SD	3.37	3.18	5.27	3.17	25.82	0.32
	n	10	10	10	10	10	10
100	Mean	16.30	15.80	14.20	3.26	10.09	22.10
	SD	3.23	3.61	3.49	8.44	8.48	0.32
	n	10	10	10	10	10	10
300	Mean	16.90	16.90	15.60	0.00	7.65	22.0
	SD	1.97	1.97	2.17	0.00	7.35	0.00
	n	10	10	10	10	10	10
1000	Mean	18.10	17.40	16.40	6.16	8.21	22.10
	SD	5.17	5.32	5.44	12.73	9.93	0.32
	n	10	10	10	10	10	10

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

Treatment (mg/kg/bw/d)		At birth			On day 1 post-partum		On day 4 post-partum
		Total litter size	Live litter size	Pup loss (%)	Litter weight (g)	Mean pup weight (g)	Live litter size	Cumulative loss (%)	Litter weight (g)	Mean pup weight (g)
0	Mean	15.60	15.40	1.13	101.62	7.00	14.40	6.37	132.38	9.61
	SD	5.27	5.19	2.40	30.0	1.09	4.60	7.58	36.25	1.58
	n	10	10	10	10	10	10	10	10	10
100	Mean	14.20	14.20	0.00	96.87	7.01	14.10	0.56	145.70	10.51
	SD	3.49	3.49	0.00	19.08	0.81	3.38	1.77	29.27	1.08
	n	10	10	10	10	10	10	10	10	10
300	Mean	15.60	15.60	0.00	106.70	6.94	14.80	5.51	143.13	9.76
	SD	2.17	2.17	0.00	13.11	0.44	2.66	5.60	22.54	0.97
	n	10	10	10	10	10	10	10	10	10
1000	Mean	16.40	16.20	5.50	115.50	7.10	15.60	13.30	163.63	9.46
	SD	5.44	5.67	15.71	41.59	0.55	5.82	30.67	22.38	0.74
	n	10	10	10	10	10	10	10	9	9

Table 4: Sex ratio of pups – Group mean data

Treatment (mg/kg/bw/d)		At birth				On day 4 post-partum
		Males	Females	Total	% Males	Males	Females	Total	% Males
0	Mean	6.90	8.70	15.60	49.67	6.40	8.00	14.40	49.66
	SD	2.02	4.03	5.27	19.96	1.71	3.59	4.60	19.93
	n	10	10	10	10	10	10	10	10
100	Mean	6.90	7.30	14.20	50.50	6.80	7.30	14.10	50.14
	SD	1.79	2.95	3.49	15.38	1.81	2.95	3.38	15.72
	n	10	10	10	10	10	10	10	10
300	Mean	6.20	9.40	15.60	39.23	5.90	8.90	14.80	39.36
	SD	2.20	1.65	2.17	11.28	2.08	1.66	2.66	10.29
	n	10	10	10	10	10	10	10	10
1000	Mean	9.10	7.30	16.40	55.58	9.56*	7.78	17.33	55.66
	SD	3.57	3.62	5.44	12.47	2.24	2.86	2.06	12.97
	n	10	10	10	10	9	9	9	9

*= mean value of group is significantly different from control 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises adequate and reliable (Klimisch scores ≤2) studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on common precursors and breakdown products of hydrolysis and consistent trends in environmental fate, eco-toxicological and toxicological profile. 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 fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Read across justification

There are no data on the reproduction toxicity of Lauryl nonanoate (CAS 17671-26-0). 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).

Toxicity to reproduction

CAS 22393-85-7

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed according to OECD Guideline 422 under GLP conditions (key study, 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 oral 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, pre-coital interval, and number of corpora luteae and implantation sites, gestation length) were observed, compared with the control animals. The testis weight, epididymis weight, and histological examination of the testes in males as well as the weight and histological examination of the uterus and ovaries in females did not reveal any substance-related effects in the parental animals. All the pregnant females gave birth to live pups with the exception of one high dose female which had a total litter loss on day 3 post-partum. This female and two females in the control group had unilateral implantation, which is not considered to be treatment-related.

Therefore, a NOAEL for parental fertility of≥ 1000 mg/kg bw/day was derived for male and female rats.

 

Overall conclusion for effects on fertility

Analogue read-across from source substances was applied for reproduction toxicity. No effects on reproductive parameters/organs were observed in the available screening study. The NOAEL for reproduction toxicity was at the limit dose of 1000 mg/kg bw/day. Based on the available data and following the analogue approach, absence of adverse effects on fertility can be anticipated for the target substance Lauryl nonanoate (CAS 17671-26-0).

Effects on developmental toxicity

Description of key information

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

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Exposure duration was only from day 6-15 of gestation instead of day 5-19, analytical purity of test substnace not specified.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 1981

Deviations:

yes

Remarks:

no analytical purity given

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 2001

Deviations:

yes

Remarks:

Exposure duration was only from day 6-15 of gestation instead of day 5-19; no analytical purity given

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 weeks
- Weight at study initiation: mean approx. 197 g
- Housing: individually in Makrolon Type M3 cages
- Diet: Pelleted Altromin Maintenance Diet 1324 (Altromin GmbH, Lage, Germany), ad libitum (analytically controlled per batch)
- Water: tap water, ad libitum (once weekly controlled)
- 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: Arachis oil, DAB 10

Details on exposure:

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

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw

All groups received a dose volume of 5 mL/kg body weight, adjusted to the body weight of day 6 post coitum.

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

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.

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
- Other: dead/living foetuses

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) The Wilson technique was applied to half of the foetuses to evaluate potential visceral changes (Wilson and Warkany, 1965).
2) The remaining fetuses were placed non individually in a solution of potassium hydroxide for clearing and were stained with alizarin red according to Dawson, 1926. 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.
- 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).

Historical control data:

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

Clinical signs:

no effects observed

Description (incidence and severity):

The dams tolerated the applied dose levels of up to 1000 mg/kg bw/day without lethality and clinical signs of systemic toxicity.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

The dams tolerated the applied dose levels of up to 1000 mg/kg bw/day without lethality and clinical signs of systemic toxicity.
No death occurred in the dams of group 1 (vehicle control) and in the test groups 2 - 4.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Maternal body weight gain was not affected by the treatment. 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 consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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

Gross pathological findings:

no effects observed

Description (incidence and severity):

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

Neuropathological findings:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

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.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre- and postimplantation loss and mean numbers of resorption were unaffected by treatment. All parameters were comparable with the animals of the control group.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

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.

Early or late resorptions:

no effects observed

Description (incidence and severity):

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.

Dead fetuses:

no effects observed

Description (incidence and severity):

Apart from dose group 1000 mg/kg bw/day (one dead foetus) all females had viable foetuses.

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

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

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio of the foetuses was not affected by the treatment with the test substance.

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

No macroscopical findings were noted at external examination of foetuses 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 foetus with paleness and one dead foetus.

Skeletal malformations:

no effects observed

Description (incidence and severity):

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.

Visceral malformations:

no effects observed

Description (incidence and severity):

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.

Details on embryotoxic / teratogenic effects:

No 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.

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

Remarks:

embryotoxicity/teratogenicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects on: litter size and weights; number viable (number alive and number dead); sex ratio; number of implantations; no. of total litter losses by resorption.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

2 -ethylhexyl stearate 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 1000 mg/kg bw/day.

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable (Klimisch scores 2) study from a reference substances with similar structure and intrinsic properties. Read-across is justified based on common precursors and breakdown products of hydrolysis and consistent trends in environmental fate, eco-toxicological 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

There are no data on the developmental toxicity of the target substance lauryl nonanoate (CAS 17671-26-0). The assessment was therefore based on studies conducted with analogue source 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).

CAS 22047-49-0

A developmental toxicity study was performed according to OECD Guideline 414 under GLP conditions (key study, 2000). 24 female Sprague-Dawley rats per dose were administered 0, 100, 300 and 1000 mg/kg bw/day 2-ethylhexyl stearate (CAS 22047-49-0) once daily from day 6 up to day 15 of gestation via gavage in arachis oil as vehicle.

The dams tolerated the applied dose levels of up to 1000 mg/kg bw/day without lethality and clinical signs of systemic toxicity. No treatment-related adverse effects on body weight, behaviour, gross pathology, number of abortions, pre- and post-implantation loss, total litter loss, organ weights, fetal body weight, sex ratio, external, visceral and skeletal malformations were found. 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 lack of adverse toxic effects, the NOAEL for developmental toxicity/teratogenicity and for maternal developmental toxicity was considered to be ≥ 1000 mg/kg bw/day.

CAS 22393-85-7

A combined repeated dose toxicity study with a reproduction/developmental toxicity screening test was performed according to OECD Guideline 422 under GLP conditions (key study, 2014) with Tetradecyl oleate (CAS 22393-85-7). No toxicologically relevant treatment related adverse effects were seen on viability, clinical signs, body weight, sex ratio, and gross pathology of F1 offspring. Based on these results, a developmental NOAEL of ≥ 1000 mg/kg was determined.

Overall conclusion for developmental toxicity/teratogenicity

Analogue read-across from source substances was applied for developmental toxicity. Based on the available data and following the analogue approach, absence of developmental toxicity can be anticipated for the target substance Lauryl nonanoate (CAS 17671-26-0).

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 Lauryl nonanoate (CAS 17671-26-0), 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.

Therefore, based on the analogue read-across approach, the available data on reproduction toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.