Fatty acids, C16-18, isononyl esters

Endpoint summary

Description of key information

Oral (OECD 422, read across): NOAEL female rat = 300 mg/kg bw/day

Oral (OECD 422, read across): NOAEL male rat = 1000 mg/kg bw/day

Oral (OECD 407, read across): NOAEL, male and female rat >1000 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

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

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

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:

Remarks:

Source: CAS 59231-34-4

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Source: CAS 59231-34-4

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Source: CAS 91031-48-0

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Source: CAS 3687-46-5

Key result

Critical effects observed:

no

Conclusions:

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

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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 oral exposure:

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

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)

Observations and examinations performed and frequency:

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily by visual appraisal

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the pre-mating period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, overnight
- How many animals: 5/sex/dose
- Parameters: haemoglobin, erythrocytes, leucocytes, differential blood count (absolute/relative), reticulocytes, platelets, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, thromboplastin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the pre-mating period
- Animals fasted: Yes, overnight
- How many animals: 5/sex/dose
- Parameters examined: albumin, globulin, albumin/globulin ratio, bile acids, bilirubin, cholesterol (total), creatinine, glucose, urea, total protein, calcium, chlorid, potassium, sodium, alanine aminotransferase (ALAT), alkaline phosphatase (AP), aspartate aminotransferase (ASAT)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: two hours after dosing shortly before scheduled sacrifice in 5 males per group (day 35); two hours after dosing during lactation, shortly before scheduled sacrifice in 5 females per group (day 39-56)
Screening of assessment were conducted as described on the following pages in five males and five females randomly selected from each group.
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) (based on Gad), as well as the assessment of grip strength (Meyer) and motor activity

OTHER: Qualitative sperm analysis was performed.
(for further reproduction parameters see respective study entry (chapter 7.8.1))
Males were sacrificed on day 36, females were sacrifices on day 4 post-partum or shorty thereafter.

Sacrifice and pathology:

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)

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.

Clinical signs:

no effects observed

Description (incidence and severity):

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

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

statistically significant decrease (-9.4%) in body weight in females during lactation at 1000 mg/kg bw/day

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the high dose group (females: gestation/lactation period) a statistically significant reduction in food consumption by 21.7% was noted.

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

Description (incidence and severity):

An decrease of ASAT activity was seen in high dose females on day 15. This effects was considered to be due to the relative low or high value observed for the control group and not to be test item related.

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

Description (incidence and severity):

All effects observed (slight increase in absolute and relative liver weight in males) were still within the historical control data of the laboratory and thus not of toxicological relevance.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

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).

Key result

Dose descriptor:

NOAEL

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

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

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

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

16 Nov 1982 - 19 Jan 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

No monitoring of clinical signs outside the home cage, no neurobehavioural examinations.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

no monitoring of clinical signs outside the home cage, no neurobehavioural examinations

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: Wistar Han.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Züchter Zentralinstitut für Versuchstiere, Hannover, Germany
- Age at study initiation: approximately 4 weeks
- Weight at study initiation: 50-78 g (males); 50-82 g (females)
- Housing: 2-3 animals/sex/cage in Macrolon cages (type III) with wood shavings (Arwi-Center, Essen, Germany)
- Diet: Altromin 1324 DK pelleted, nitrosamine-poor diet (Fa. Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (ºC): 21-22
- Humidity (%): 49-65
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The dosing solutions were prepared daily prior to administration. The application volumes were adapted weekly to the current body weights.

VEHICLE
- Concentration in vehicle: 2, 10 and 20% in olive oil for the 100, 500 and 1000 mg/kg bw/day groups, respectively
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days (21 doses) and 28 days post-exposure observation period (satellite control and treated groups)

Frequency of treatment:

Daily, 5 days/week

Dose / conc.:

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

Dose / conc.:

500 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 (main groups, control and all dose levels)
5 (satellite groups, control and all dose levels)

Control animals:

yes, concurrent vehicle

Details on study design:

- Post-exposure recovery period in satellite groups: 28 days. A satellite groups was included in the control group and each dose group.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations examined: mortality, clinical signs

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION : Yes. Determined per cage per week.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes. Determined per cage per week.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to necropsy
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Yes, ether
- Animals fasted: No data
- How many animals: all dose groups
- Parameters examined: haematocrit, haemoglobin content, red blood cell count, white blood cell count, mean cell volume, thrombocyte cell count, differential blood count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: No data
- How many animals: all dose groups
- Parameters examined: urea, creatinine, glucose, sodium, potassium, calcium, inorganic phosphorus, alkaline phosphatase, serum alanine aminotransferase, serum aspartate aminotransferase, gamma glutamyl transpeptidase, chloride, albumin, total protein, cholesterol

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the study period
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: pH, proteins, glucose, blood cells, bacteria, leucocytes, erythrocytes, urea, sodium oxide, urobilin, ket., specific gravity, epi., trip.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: faeces were collected at the end of the study and examined for parasites, appearance and amount.

Sacrifice and pathology:

GROSS PATHOLOGY:
Yes. Gross pathology was performed in all animals, including the satellite group animals. The absolute and relative weight of the following organs was determined: thyroid, adrenals, thymus, spleen, heart, kidneys, brain, testes, ovaries and liver.

HISTOPATHOLOGY:
Yes, in all animals in the control and 1000 mg/kg bw/day groups the following organs were examined: brain (cerebrum, cerebellum), eye, tongue, salivary glands, auxillary lymph nodes, oesophagus, trachea, heart, aorta, lungs, bronchial lymph nodes, liver, spleen, kidneys,
urethra, pancreas, thymus, thyroid, parathyroid, adrenals, peripheral nerve, forestomach, stomach, duodenum, jejunum, ileum, colon, caecum, rectum, anal mucosa, Peyer's patches, mesenterial lymph node, gall bladder, urinary bladder, vagina, uterus, ovary, prostate, testes, epididymes, prostate + seminal vesicles with coagulating glands, skin, muscle, skin lymph nodes, mammary gland.

The target organ of the main groups was the liver, which was examined in greater detail in 3 male and 3 female animals in the control and 1000 mg/kg bw/day groups. In the satellite groups, the target organs (lungs, pancreas, maxillary lymph node, salivary gland and forestomach) were examined in 3 females and males from the control and 1000 mg/kg bw/day satelllite groups.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight was statistically significantly lower in the male 100 mg/kg bw/day group in week 0 and 1. As the rats weighed, on average, less from the start of the study, this reflects a mistake in standardising the group and is not related to the treatment (see Table 1).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The food consumption in females and males administered 1000 mg/kg bw/day was statistically significantly increased in week 2, while in the males in this dose group the intake was reduced in week 3. Both males and females in the 500 mg/kg bw/day group had a statistically significant increase in food consumption in week 2. These effect were transient and were considered not to be treatment-related (see Table 1).

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

The water intake was statistically significantly increased in males in the highest dose group in week 2. For males administered 100 mg/kg bw/day the intake was decreased in week 1 and 2. In the female groups, the water consumption was reduced with statistical significance in females administered 100, 500 and 1000 mg/kg bw/day in week 2, week 3 and 4, and week 3, respectively. These effect were transient and were considered not to be substance-related (see Table 1).

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant decreases in haemoglobin levels were observed in all dose groups. As the changes were not dose-related and no other relevant effects were seen in histopathological parameters, this is not considered to be a toxicologically relevant effect. A reduction in the haematocrit level and red blood cell count in the 500 mg/kg bw/day females was considered to be incidental as the effect was only observed at one dose level and in one sex.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The inorganic phosphorus level was increased with statistical significance in all male dose groups and in the 100 and 500 mg/kg bw/day female group. The effect was not dose-related in the males and not observed at the highest dose level in females, therefore it was not considered to be substance-related. The decrease in the alkaline phosphatase level in females in the 500 mg/kg bw/day group was deemed to be incidental and not substance-related.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In females administered 1000 mg/kg bw/day, the absolute kidney weight was increased. The relative weight was not affected and the change was seen only in one sex, leading to the conclusion that this effect had no toxicological relevance. The relative heart weight in males administered 500 and 1000 mg/kg bw/day was reduced slightly, but with statistical significance, while the absolute heart weight was increased in the 500 mg/kg bw/day group. The highest male dose group was not affected and no related effects were noted in the histopathological examination. Therefore, this effect was considered of no toxicological relevance. Some statistically significant changes in organ weights of the thyroid, spleen and adrenal glands were noted in males or females in the 100 or 500 mg/kg bw/day groups. As the changes were either absolute or relative weight, only in one sex and not observed in the highest dose level, they were not considered to be treatment-related. No treatment-related changes were noted in the salivary gland, lungs, pancreas or stomach in the satellite animals.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No substance-related effects were observed on organs or tissues during the histopathological examination. The mucosa of the forestomach in control and treated groups showed degenerative changes or signs of inflammation. 2/10 males and 3/10 females in the control groups plus 2/10 females in the 1000 mg/kg bw/day group had (suspected) hyperplasia; and 7/10 control males, 7/10 control females, 5/10 1000 mg/kg bw/day males and 7/10 1000 mg/kg bw/day females showed focal eosinophilic infiltration of the forestomach. These effects were possibly caused by the repeated use of a stomach tube for the dosing by gavage. As humans do not have a forestomach, this effect is not relevant to human exposure.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Eggs from a parasite were detected in the faeces of 1 female in the high-dose group. No other significant effects were seen on the faeces appearance or amount.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Table 1: Data of selected parameters determined in the study for all groups:

Food consumption (g/animal/d), group mean values
week	group***, sex (m/f)
	1 m	2 m	3 m	4 m	1 f	2 f	3 f	4 f
1	18	16	18	18	14	15	14	15
2	19	18	21**	22**	14	14	15**	16*
3	22	21	21	21*	16	17	15	16
4	17	17	19	19	13	13	13	14
Water consumption (mL/animal/d), group mean values
week	group, sex (m/f)
	1 m	2 m	3 m	4 m	1 f	2 f	3 f	4 f
1	29	25*	27	29	27	24	24	24
2	28	25**	30	31*	28	22**	26	27
3	33	29	30	30	32	27	26*	25*
4	30	25	27	29	29	28	24*	24
Body weights and weight gain (g)
week	group, sex (m/f)
	1 m	2 m	3 m	4 m	1 f	2 f	3 f	4 f
0	67	60*	64	67	64	63	60	63
1	107	96**	105	108	96	96	94	95
2	147	137	147	150	123	124	122	124
3	189	178	191	196	146	147	146	147
4	220	208	227	229	161	162	163	173
gain week 0-4	153	148	163	162	97	99	103	110
Selected haematology and clinical chemistry parameters (week 4)
	group, sex (m/f)
	1 m	2 m	3 m	4 m	1 f	2 f	3 f	4 f
haemoglobin mmol/L	10.9	10.1**	9.9**	9.8**	10.6	9.9**	9.9**	9.8**
red blood cell count T/L	7.2	6.9	6.6	6.8	6.9	6.4	6.0*	6.5
haematokrit l/L	0.39	0.38	0.36	0.37	0.36	0.34	0.32**	0.35
alkaline phosphatase U/L	371	356	390	361	247	211	196*	206
inorganic phosphate	3.1	2.9*	2.7**	2.8*	2.6	2.4*	2.4	2.5
Organ weights after 4 weeks
	group, sex (m/f)
	1 m	2 m	3 m	4 m	1 f	2 f	3 f	4 f
thyroid absolute (mg)	25	22*	23	23	21	23	19	22
spleen absolute (g)	0.57	0.59	0.63*	0.63	0.46	0.45	0.46	0.46
heart absolute (g)	0.80	0.73*	0.78	0.80	0.61	0.61	0.58	0.64
kidney absolute (g)	1.66	1.58	1.73	1.80	1.19	1.22	1.19	1.28*
thyroid relative (% of body weight)	0.010	0.010	0.009*	0.009	0.013	0.014	0.011	0.013
adrenal relative (% of body weight)	0.025	0.025	0.024	0.025	0.046	0.044	0.039*	0.045
heart relative (% of body weight)	0.33	0.32	0.31**	0.31*	0.36	0.36	0.34	0.37

* level of significance 95% in comparison with control value

** level of significance 99% in comparison with control value

*** 1 = control, 2 = 100 mg/kg bw/day, 3 = 500 mg/kg bw/day, 4 = 1000 mg/kg bw/day

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

(analytical purity of test substance not specified; no urine and neurobihavioural examinations)

Qualifier:

according to guideline

Guideline:

other: 87/302/EWG, Annex, Part B

Deviations:

yes

Remarks:

analytical purity of test substance not specified; no urine and neurobihavioural examinations

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

analytical purity of test substance not specified; no urine and neurobihavioural examinations

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany.
- Age at study initiation: ca. 40 days
- Weight at study initiation: 143 - 168 g (153.4 g mean body weight)
- Housing: 2-3 animals per Makrolon Type III cage on softwood bedding (ARWI-Center, Essen, Germany).
- Diet: pelleted Altromin 1324, Altromin, Lage, Germany, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 48-60
- Air changes (per hr): nodata, 80-490 Lux (the cages were rotated weekly in the rack)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: March 28th, 1989 - April 28th, 1989

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The application volume was 5 mL/kg bw.
The dosing solutions were prepared daily immediately before application.
The following concentrations were prepared: 100 mg/ kg bw /day: 2%; 500 mg/kg bw/day: 10%, 1000 mg/kg bw/day: 20%
The total number of applications was 23 or 24 (depending on the day of necropsy).

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 5 days/week

Dose / conc.:

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

Dose / conc.:

500 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:

- Rationale for selecting satellite groups: Additionally 5 male and 5 female animals per dose were dosed with 0 and 1000 mg/kg/day to determine the reversibility of possible compound-related findings (recovery group).
- Post-exposure recovery period in satellite groups: 27 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day, 5 days a week for mortality and clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at study termination
- Dose groups that were examined: control group and highest dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination
- Anaesthetic used for blood collection: not reported
- Animals fasted: No data
- How many animals: all animals
- Parameters checked: RBC, HCT, MCV, HGB, WBC, PLT, differential blood count (banded neutrophils, segmented neutrophils, lymphocytes, eosinophils, monocytes, basophils, myelocytes).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination
- Animals fasted: No data
- How many animals: all animals
- Parameters checked: Urea, Creatinine, Sodium, Potassium, Glucose, Calcium, ASAT, ALAT, AP, gamma-GT, Bilirubin, Chloride, total protein, total Cholesterol.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

ORGAN WEIGHTS:
Absolute and relative organ weights were determined for the following:
Brain, testes, heart, liver, spleen, adrenals, kidneys, thymus.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals
HISTOPATHOLOGY: Yes, animals of the control and highest dose group.
Following organs were examined microscopically:
Aorta thoracica, eyes, large and small intestinum, glandular stomach, cerebrum, urinary bladder, skin, heart, testes, pituitary, cerebellum, liver, trachea, lung, maxillary lymph nodes, mesenterial lymph nodes, spleen, epididymides, adrenal, peripheral nerv, kidney, ovaries, pancreas, prostate, vesicular gland, thymus, salivary gland, oesophagus, sceletal muscle, thymus, uterus, stomach, tongue.

Statistics:

T-tests according to Sachs and Dunnett; steel-test

Clinical signs:

no effects observed

Description (incidence and severity):

No symptoms were noted as compound-related effects.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

The mean water intake of the male groups 2 and 4 showed a slight increase which was not considered to be compound-related.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

Some observations like hydrometra, hydronephrosis, discolouration of the thymus and one suspicion of hydrocephalus internus were considered to be spontaneous.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

The histological examination of the organs of the recovery group was not performed, because in the main groups no target organ was evaluated.

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

Key result

Critical effects observed:

no

Conclusions:

According to the described study, a daily administration of 2-Ethylhexylstearate up to 1000 mg/kg bodyweight/day for 28 days caused no cumulative-systemic toxicity to rats. The NOAEL was found to be 1000 mg/kg bw/day.

Executive summary:

2-Ethylhexylstearate was tested for systemic toxicity at repeated doses of 0 (group 1), 100 (group 2), 500 (group 3) and 1000 (group 4) mg/kg body weight/day. The compound was administered daily by gavage over a period of 28 days. 10 male and 10 female rats were used for each dose. In addition to the groups 1 and 4, 5 male and 5 female animals were used to determine the reversibility of possible compound-related findings (recovery group).

 

All doses applied were tolerated without lethality. No symptoms were noted as compound-related effects. The mean food consumption of all groups was comparable to the control. The mean water intake of the male groups 2 and 4 showed a slight increase which was not considered to be compound-related. The total body weight gain was comparable to control in all male and female test groups. The haematological examinations showed no compound-related effects. The clinical chemistry showed no compound-related effects. The examination of the eyes by slit lamp microscope showed no compound-related effects. The absolute and relative organ weights showed no compound-related effects. The macroscopical examination of the organs displayed no compound-related effects. Some observations like hydrometra, hydronephrosis, discolouration of the thymus and one suspicion of hydrocephalus internus (animal No. 67) were considered to be spontaneous. The histological examination of the organs of all groups displayed no compound-related effects. The histological examination of the organs of the recovery group was not performed, because in the main groups no target organ was evaluated.

Endpoint conclusion

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 1-2) 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, 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, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Read Across Justification

There are no data on the repeated dose toxicity of 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 analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

Repeated dose toxicity, oral, subacute

CAS 59231-34-4

A combined repeated dose toxicity and reproduction/developmental toxicity screening study was performed according to OECD guideline 422 and under GLP conditions (key study, 2013) with Isodecyl oleate (CAS 59231-34-4). 10 rats/sex/dose were administered 0, 100, 300 and 1000 mg/kg bw/day once daily for 35 days (males) and up to 56 days (females) via oral gavage. The application started two weeks before mating and ended on the day of or one day before sacrifice. Day of sacrifice was on day 35 for the male rats and on lactation day 3 or shortly thereafter for the female rats. There was no mortality during the study period. No toxicologically relevant clinical signs were observed. A statistically significant decrease in body weight (‑9.4%) and food consumption (‑21.7%) was noted in females at 1000 mg/kg bw/day during lactation. There were no toxicologically relevant effects on water consumption and organ weights. No treatment-related effects were noted on the ophthalmology -, haematology - and clinical biochemistry parameters. The macroscopic examination at autopsy and subsequent histopathological examination did not reveal any treatment-related changes. The NOAEL for systemic toxicity was considered to be 300 mg/kg bw/day for female rats and 1000 mg/kg bw/day for male rats.

CAS 91031-48-0

A 28-day oral repeated dose toxicity study was performed with Fatty acids, C16-18, 2-ethylhexyl esters (CAS 91031-48-0) similar to OECD 407 and under GLP conditions (supporting study, 1992). Ten Sprague-Dawley rats per sex and dose were administered once daily (5 days a week) 0, 100, 500 and 1000 mg/kg bw/day by gavage, for 28 consecutive days. There was no mortality and no toxicologically relevant clinical signs were observed during the study period. No significant differences in body weight, body weight gain, food consumption, water consumption and organ weight were noted between the control group and treatment groups. No treatment-related effects on the ophthalmology-, hematology- and clinical biochemistry parameters were observed. No treatment-related changes were noted during the gross pathology and histopathology examinations. Based on the absence of adverse toxic effects the NOEL for systemic toxicity was 1000 mg/kg bw/day.

CAS 3687-46-5

A 28-day oral repeated dose toxicity study was performed with Decyl oleate (CAS 3687-46-5) similar to OECD 407 (supporting study, 1987). Ten Wistar rats per sex and dose and 5 per sex and dose satellite rats were administered once daily (5 days a week) 0, 100, 500 and 1000 mg/kg bw/day by gavage, for 28 consecutive days. There was no mortality and no toxicologically relevant clinical signs were observed during the study period. No significant differences in body weight, body weight gain, food consumption, water consumption and organ weight were noted between the control group and treatment groups. No treatment-related effects on the ophthalmology-, hematology- and clinical biochemistry parameters were observed. No treatment-related changes were noted during the gross pathology and histopathology examinations. Based on the absence of adverse toxic effects the NOAEL for systemic toxicity was 1000 mg/kg bw/day.

Overall conclusion for repeated dose toxicity

The data for the source substance showed no toxicologically relevant effects up to and including the recommended limit values. Therefore, as the available data did not identify any hazard for repeated dose toxicity, Fatty acids, C16-18, isononyl esters (CAS 91031-57-1) is not considered to be hazardous following repeated exposure.

 

In order to fulfil the standard information requirements according to Regulation (EC) No. 1907/2006, Annex IX, Column 1, Section 8.6.2, a GLP-compliant oral subchronic toxicity study following OECD guideline 408 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.

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