Fatty acids, C16-18, isononyl esters

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral: OECD 422, rat, read across, NOAEL fertility 300 mg/kg/day for females and 1000 mg/kg/day for males

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Nov - 13 Dec 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(22 Mar 1996)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Behoerde fuer Soziales, Familie, Gesundheit und Verbraucherschutz; Hamburg, Germany

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Research Models and Services Germany GmbH, Sulzfeld, Germany
- Age at study initiation: males: 50 days; females: 60 days
- Mean weight at study initiation: males: 248.8 to 298.7 g; females: 195.2 to 228.1 g
- Fasting period before study: no
- Housing: single housing in MAKROLON cages (type III plus)
- Diet: commercial ssniff R-Z V1324 (ssniff Spezialdiäten GmbH, Soest, Germany); ad libitum
- Water: tap water; ad libitum
(Analyses of diet and water was performed.)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

VEHICLE
- Concentration in vehicle: 50, 150 and 500 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1 male and 1 female animal were placed in one cage during the dark period
- 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:

For the analysis of the test item-vehicle mixtures samples were taken at the following time points and stored at ≤ -20°C until analysis:
Start of treatment period; immediately after preparation of the test item-vehicle mixtures; 8 and 24 hours after storage of the test item preparations at room temperature; end of treatment period; during treatment with the test item always before administration to the last animal of the dose level group
The following parameters were determined: linearity, accuracy, precision, sensitivity, specificity, stability at +2°C to +8°C or -20°C (0, 24, 72 and 168 hours)

Duration of treatment / exposure:

Males: The daily administration of the test item was started two weeks before mating and lasted until test day 35, which was one day before sacrifice.
Females: The daily administration of the test item was started two weeks before mating and continued to at least day 3 of lactation.
Maximum: 56 days of treatment

Frequency of treatment:

once daily; 7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on a 14-day range-finding study (Leuschner, 2012)

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before and after dosing
- Cage side observations checked: skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns

MORTALITY AND CLINICAL SIGNS
- Time schedule: at least once daily (the frequency was increased when signs of toxicity were observed); deaths were recorded twice daily (animals which died or were sacrificed during the study were necropsied as soon as possible after exitus)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure (to allow within-subject comparisons) and once a week thereafter
- Paramters: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern); changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards)

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

For further systemic effects (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

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight, and qualitative sperm staging

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of all pups/litter

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 36]
- Maternal animals: All surviving animals [females were sacrifices on day 4 post-partum or shorty thereafter]

GROSS PATHOLOGY: Yes
- Organ weights: epididymes and testicles (all males); adrenal gland, brain, heart, kidney, liver, spleen, thymus (5/sex/dose)
- Fixation: epididymis, gross lesions, mammary gland, ovary, prostate, seminal vesicle, testicle, uterus (incl. cervix and oviducts), vagina (all animals); adrenal gland, bone marrow (os femoris), brain (cerebrum, cerebellum, brain stem), heart (left and right ventricle, septum), intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method), intestine, large (colon, rectum), kidney and ureter, liver, lungs (with mainstem bronchi and bronchioles), preserved by inflation with fixative and then immersion, lymph node (1 cervical, 1 mesenteric), nerve (sciatic), oesophagus, spinal cord (3 sections), spleen, stomach, thyroid (incl. parathyroids), thymus, tissue masses or tumours (incl. regional lymph nodes), tongue (incl. base), trachea (incl. larynx), urinary bladder (5/sex/dose)
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.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

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:

STUDENT's t-test (p ≤ 0.01): all numerical functional tests
Multiple t-test based on DUNNETT (p ≤ 0.05 and p ≤ 0.01): body weight, food consumption, haematology, clinical chemistry, absolute and relative organ weights
For all numerical values homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group. In case of heterogeneity of variances, the STUDENT's t-test was carried out; limit of significance was p ≤ 0.01.

Reproductive indices:

For each group the gestation index was determined:
- Fertility Index female [%] = Number of pregnant rats/Number of females used x 100
- Gestation Index [%] = Number of litters with live pups/Number of pregnant rats x 100

Offspring viability indices:

For each litter and group the following indices were determined:
- Birth Index [%] = Total number of pups born (live + dead)/Number of implantation scars x 100
- Live Birth Index [%] = Number of pups born alive on day 0/1 Total number born (live + dead) x 100
- Viability Index [%] = Number of pups alive on day 4/Number of pups live on day 0/1 x 100
- Pre-implantation loss [%] = Corpora lutea - implantations/Corpora lutea x 100
- Post-implantation loss [%] = Implantations - number of pups born alive/Implantations x 100

Clinical signs:

no effects observed

Description (incidence and severity):

Piloerection was seen in 1 female of the high dose group at on day 2-4 of lactation.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduction in body weight (-9.4%) in high dose females during lactation period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduction in food intake (-21.7%) in high dose females during gestation/lactation.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Description (incidence and severity):

- Pre-coital time: No effects observed
- Gestation length: No effects observed

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The qualitative sperm staging revealed no test item-related specific spermatogenic changes in the male animals from the high dose group (1000 mg/kg b.w./day).

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Reproduction parameters of the dams: statistically significant increase in post implantation loss, non-statistically significant decrease in birth index, elevated number of stillbirths, leading to a statistically significant reduction in the live birth index in highest dose group (1000 mg/kg bw/day).

No effects were found for the fertility index, the gestation index and the preimplantation loss.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: adverse effects on body weight and body weight gain and food consumption at 1000 mg/kg bw/d

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: an increase in post-implantation loss, a decrease in the birth index and a decrease in the live birth index at 1000 mg/kg bw/d

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no spermatogenic changes

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

A test item-related decrease in the viability index was noted in the high dose group (1000 mg/kg bw/day) (71.3 % in the high dose group vs. 98.7% in the control group). The high number of dead pups in the high dose group was due to 2 dams with no surviving pups on lactation day 4 (deaths partly due to cannibalization). The total litter loss in 2 of 7 dams (28.6%) was considered as test item related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A non-statistically significant reduction in mean litter weight on lactation day 1 by 17.2% was noted in the high dose group (high dose group), which is regarded to be test item-related. Total litter weight was also reduced by 24.4% in comparison to controls.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

other: effects on litter weight at 1000 mg/kg bw/day

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

no

Individual body weights of females during the pre-mating and lactation period.

Control group	Day(s) relative to start
	1	8	15
11	196	209	212
12	217	228	243
13	210	220	213
14	217	234	244
15	3211	215	232
16	195	208	197
17	205	224	239
18	212	238	233
19	224	236	259
20	200	218	214
Mean	209	223	229
SD	9	10	19

1000 mg/kg bw	Day(s) relative to start
	1	8	15
71	200	210	225
72	210	231	234
73	206	234	247
74	210	222	235
75	194	219	218
76	218	243	223
77	214	230	227
78	222	225	210
79	198	200	201
80	203	223	231
Mean	207	224	225
SD	9	12	13

Control group	Day(s) relative to littering
	1	4
11	286	309
12	323	330
13	325	311
14	331	327
15	311	322
16	282	302
17	309	327
18	312	328
19	315	331
20	300	329
Mean	309	322
SD	16	10

1000 mg/kg bw	Day(s) relative to littering
	1	4
71	270	281
72	339	301
73	289	309
75	289	317
77	253	247
78	280	271
79	290	296
80	293	308
Mean	288	291
SD	24	23

Relative food consumption of females between day 1 and 4 of lactation

Control group		1000 mg/kg bw
11	122	71	115
12	91	72	96
13	105	73	63
14	107	75	110
15	106	77	30
16	121	78	63
17	114	79	98
18	100	80	110
19	101
20	126
Mean	109	Mean	86
SD	11	SD	30

Viability index [%], day 1 to 4 of lactation

Control group		1000 mg/kg bw
11	93	71	93
12	100	72	100
13	100	73	0
14	93	74	not pregnant
15	100	75	100
16	100	76	not pregnant
17	100	77	0
18	93	78	no viable pubs
19	89	79	100
20	100	80	100
Mean	96	Mean	70
SD	4	SD	48

Summary of Live Birth Index, Pre-implantation loss, and Post-implantation loss in female animals

	Control group	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Live Birth Index
Mean	100	100	100	86
SD	0	0	0	35
Total %^1	100	100	100	91
Pre-implantation loss
Mean	2.4	18.3	0.6	8.1
SD	3.1	26.0	2.0	9.8
Total %^2	2.5	20.3**	0.7	9.1*
Post-implantation loss
Mean	7.6	9.0	7.5	20.8
SD	10.5	14.8	8.8	32.9
Total %^3	7.6	10.2	7.7	21.7**

*:p < 0.05 / **: p < 0.01, Chi²-test

#1: based on the total number of live born pups and the total number of pups at birth (alive and dead)

#2: based on the total number of corpora lutea and the total number of implantation sites

#3: based on the total number of implantation sites and the total number of live born pups

 

 

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Refer to analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Source:

Remarks:

CAS 59231-34-4

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Source: CAS 59231-34-4

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Remarks on result:

other: Source:

Remarks:

CAS 59231-34-4

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

Remarks on result:

other: Source:

Remarks:

CAS 59231-34-4

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Remarks on result:

other: Source:

Remarks:

CAS 56231-34-4

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

no

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
The available data on a suitable source substance did not show any toxic effect on reproduction. Therefore, the target substance Fatty acids, C16-18, isononyl esters is not predicted to be toxic to reproduction.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises adequate and reliable (Klimisch score 1) study 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, 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 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 the target substance Fatty acids, C16-18, isononyl esters (CAS 91031-57-1). 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 (fertility)

CAS 59231-34-4

A combined repeated dose toxicity and reproduction/developmental toxicity screening study was performed according to OECD guideline 422 under GLP conditions (key study, 2013). 10 rats/sex/dose were administered 0, 100, 300 and 1000 mg/kg bw/day 8-methylnonyl (9E)-octadec-9-enoate (CAS 59231-34-4) in corn oil once daily, via oral gavage. Males were exposed from two weeks before mating on test day one until 35 days after mating. Females were exposed for up to 56 days (starting 2 weeks prior to mating up to at least day 3 of lactation).

No treatment-related parental effects were seen on mortality, clinical signs. Reduction in body weight (-9.4%) and food intake (-21.7%) was seen in high dose females during gestation/lactation.

No effects on water consumption, ophthalmology, haematological parameters, clinical chemistry parameters, macroscopic and microscopic pathology were observed. The NOAEL for parental systemic toxicity was 300 mg/kg/day for females and ≥1000 mg/kg bw/day for males.

No effects on the reproductive function of males (qualitative sperm staging) was observed. A statistically significant increase in post implantation loss, non-statistically significant decrease in birth index, elevated number of stillbirths, leading to a statistically significant reduction in the live birth index was observed in dams of the highest dose group. These effects were regarded as secondary non-specific consequence of adverse effects on food consumption and body weight (gain) in maternal animals at 1000 mg/kg bw/day.

The NOAEL for parental fertility was 300 mg/kg/day for females and ≥1000 mg/kg bw/day for males.

Overall conclusion for effects on fertility

Analogue read-across from the source substance 8-methylnonyl (9E)-octadec-9-enoate (CAS 59231-34-4) was applied for toxicity to reproduction (fertility). Based on the available data and following the analogue approach, no hazard for reproduction toxicity was identified for the target substance Fatty acids, C16-18, isononyl esters (CAS 91031-57-1).

According to Annex IX, Section 8.7.3, Column 1, of the REACH Regulation (EC) No. 1907/2006 an extended one-generation reproductive toxicity study (EOGRTS, OECD 443) must be performed 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 to reproductive toxicity. Testing of fatty acids, C16-18, isononyl esters (CAS 91031-57-1) according to OECD 414 and OECD 408 is proposed to fulfil the standard information requirements of Annex IX, REACH Regulation (EC) No. 1907/2006. In addition, a combined repeated dose toxicity and reproduction / developmental toxicity screening study according to OECD 422 will be commissioned to obtain a valid and robust data basis for a reliable decision related to the EOGRTS. The available repeated dose toxicity as well as reproductive / developmental data for various analogue source substances do not provide any concern in relation to reproductive toxicity of fatty acids, C16-18, isononyl esters (CAS 91031-57-1). The effects observed in an OECD 422 screening study with the structural analogue 8-methylnonyl (9E)-octadec-9-enoate (CAS 59231-34-4) in dams of the highest dose group (statistically significant increase in post implantation loss, non-statistically significant decrease in birth index, elevated number of stillbirths, leading to a statistically significant reduction in the live birth index) were regarded as secondary non-specific consequence of adverse effects on food consumption and body weight (gain) in maternal animals at 1000 mg/kg bw/day. Please refer to the analogue justification provided in IUCLID section 13 for details on the analogue approach. Taken together, based on the current knowledge and planned testing strategy, no EOGRTS is required to fulfil the requirements according to Annex IX, Section 8.7.3, Column 1, of the REACH Regulation (EC) No. 1907/2006.

Effects on developmental toxicity

Description of key information

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

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

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

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): 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 (nominal)

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 (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: embryotoxicity

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Refer to analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Species:

rat

Key result

Dose descriptor:

NOAEL

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

Remarks:

Source: CAS 91031-48-0

Key result

Abnormalities:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Source: CAS 91031-48-0

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Source: CAS 91031-48-0

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
The available data on a suitable source substance did not show any adverse effect on development. Therefore, the target substance Fatty acids, C16-18, isononyl esters is not predicted to be toxic to development.

Effect on developmental toxicity: 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 score 1-2) 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 (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

Developmental toxicity/teratogenicity

CAS 59231-34-4

In a combined repeated dose toxicity and reproduction/developmental toxicity screening study performed with 8-methylnonyl (9E)-octadec-9-enoate (CAS 59231-34-4) according to OECD guideline 422, the NOAEL for developmental toxicity was found to be 300 mg/kg bw per day. This NOAEL was based on effects on the viability and litter weight of foetuses seen in the highest dose group at 1000 mg/kg bw per day. The effects were regarded as secondary non-specific consequences of adverse effects on food consumption and body weight (gain) in maternal animals at 1000 mg/kg bw/day.

 

CAS 91031-48-0

A developmental toxicity study was performed according to OECD Guideline 414 under GLP conditions (key study, 1994). This study was selected as key study for assessment of developmental toxicity of the target substance.

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.

 

Overall conclusion for developmental toxicity/teratogenicity

Analogue read-across from the source substance Fatty acids, C16-18, 2-ethylhexyl esters was applied for developmental toxicity/teratogenicity. Based on the available data and following the analogue approach, no hazard for reproduction (development/teratogenicity) was identified for the target substance Fatty acids, C16-18, isononyl esters (CAS 91031-57-1).

 

In order to fulfil the standard information requirements according to Regulation (EC) No. 1907/2006, Annex IX, Column 1, Section 8.7.2, a GLP compliant pre-natal developmental toxicity study in one species via the oral route following OECD guideline 414 is proposed. Column 2 adaption possibilities at the Annex IX level where considered and do not apply. In addition, the general adaptation possibilities of Annex XI of the REACH Regulation are not adequate to generate the necessary information.

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, C16-18, isononyl esters (CAS 91031-57-1), 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 or Directive 67/548/EEC, and is therefore conclusive but not sufficient for classification.

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