Diesters of alcohols, C7-9-iso-, C8-rich, 2-ethylhexyl and nonanedioic acid

Endpoint summary

Description of key information

In a study according to OECD 422 male and female rats were exposed to bis(2-ethylhexyl) azelate by gavage during 14 days before mating, 28 days afterwards (males) or during pregnancy until day 4 of lactation. There was no mortality and clinical signs did not differ between treated and control animals. Body weight gain was suppressed in males at 1000 mg/kg bw from day 28 onwards. No adverse effects on food consumption, haematology and behavioural assessment were seen. Clinical chemistry revealed a dose related increase in albumin/globulin (A/G) ratio in males at 1000 mg/kg bw and in females at 300 and 1000 mg/kg bw. The effect in females at 300 mg/kg bw was not accompanied by histopathological changes. Decreases in total protein, creatinine and calcium were observed in females at 1000 mg/kg bw/day. There were increases in relative weight of liver and absolute and relative weights of kidney for males and increases in relative weights of liver and kidney for females were observed in the 1000 mg/kg. For the liver histopathology a tendency of increase in hypertrophy of centrilobular hepatocytes and a reduction in the grade of periportal fatty change was observed in males of the 1000 mg/kg bw (not in females)

Based on these findings the NOAEL for parental effects is set at 300 mg/kg bw.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - 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)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crj:CD(SD)IGS

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: 10 weeks
- Housing: Metal mesh cage
- Diet: ad libitum, CE-2 from CLEA Japan, Inc., Meguro, Japan
- Water: ad libitum, tap water
- Acclimation period: about 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.0 - 25.5
- Humidity (%): 49.5 - 73.0
- Air changes: 15 times/day
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solution was prepared more than once a week and stored at room temperature in the dark for 7 days. The stability for 8 days was verified by GC.

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing solution was mixtured with n-hexane and concentrations were analyzed by GC.

Duration of treatment / exposure:

Males: 14 days before mating, 28 days afterwards
Females: total of 42-53 days beginning 14 days before mating to day 4 of lactation

Frequency of treatment:

7 days/week

Remarks:

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

No. of animals per sex per dose:

13

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily before and after treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at 13:00-16:00 on Days 7, 14, 21, 28, 35 and 42 of the treatment period using scoring systems.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were determined on Days 1, 7, 14, 21, 28, 35, 42 of treatment and prior to necropsy in males and on Days 1, 7, 14 and 21 of treatment, Days 0, 7, 14 and 20 of gestation, Days 0 and 4 of lactation and prior to necropsy in females.

FOOD CONSUMPTION: Yes
- Time schedule: Days 1-2, 7-8, 14-15, 29-30, 35-36 and 41-42 of treatment in males, and on Days 1-2, 7-8 and 14-15 of the treatment period, Days 0-1, 7-8, 14-15 and 20-21 of gestation and day 3-4 of lactation in females. Food consumption was not determined during the mating period in males and females.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from the abdominal aorta after overnight starvation on
next day of the last administration.
- Animals fasted: Yes
- How many animals: All
- Following parameters were checked: red blood cell (RBC), hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cells (WBC), neutrophils, eosinophils, basophils, monocytes, lymphocytes, platelets, prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from the abdominal aorta after overnight starvation on
next day of the last administration.
- Animals fasted: Yes
- How many animals: All
- Following parameters were checked: total protein, albumin, albumin/globulin ratio (A/G ratio), blood urea nitrogen (BUN), creatinine, glucose, total cholesterol, triglycerides, alkaline phosphatase (ALP), alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, total bilirubin, inorganic phosphor, Ca, Na, K, Cl

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on Day 42 of treatment in males and on Day 4 of lactation in females.
- Dose groups that were examined: 5 animals/sex/dose
- Battery of functions tested: Preyer's reflex, pupillary reflex, pain reaction, withdrawal reflex, eyelid reflex and righting reflex

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Organ weights: Brain, heart, liver, kidney, spleen, adrenal, thymus, testis and epididymis
HISTOPATHOLOGY: Yes
Testis, epididymis and ovary of all animals at all doses, and
brain, pituitary, thymus, thyroid, parathyroid, adrenal, spleen, heart, stomach,
liver, duodenum, jejunum, ileum, cecum, colon, rectum, trachea, lung, kidney, urinary
bladder, femur, spinal cord, mesentery lymph node, submandibular gland, sciatic nerve,
bone marrow, prostate, seminal vesicle, uterus and vagina of 5 males and females at 0
and 1000 mg/kg bw/day.

Statistics:

Statistical methods: Dunnett's test for continuous data, and Mann-Whitney' U-test and Fisher' test for discrete data.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: impaired BW gain in males

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw: decrease in white blood cells

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: increase in albumin/globulin ratio in males/females, decrease in total protein and Ca in females; 300 mg/kg bw/day: increase in albumin/globulin ratio in females

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day group: increase in rel. liver and kidney weight in males and females (non adverse)

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw:/day: hepatocellular hypertrophy and periportal fatty change in males, (non adverse)

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: There was no mortality related to the test substance treatment. No changes were observed on general clinical observation, nor were scores obtained by detailed clinical observations between control and the test substance-treated groups.

BODY WEIGHT AND WEIGHT GAIN
Body weight gain was suppressed in males of the 1000 mg/kg bw/day-treated group (see Table 1)

FOOD CONSUMPTION
No effects were observed in males and females of the test substance-treated groups.

HAEMATOLOGY
No effects were observed in males of the test substance-treated groups. A decrease in white blood cells (WBC) and a shorter activated partial thromboplastin time were observed in females of the 1000 mg/kg bw/day dose group. But these change were not considered as adverse effects because of no accompanying changes. A decrease in WBC was observed in females of the 1000 mg/kg bw/day dose group.

CLINICAL CHEMISTRY
An increase in albumin/globulin (A/G) ratio was found in males at 1000 mg/kg bw/day and in females at 300 mg/kg bw/day and higher. Decreases in total protein, creatinine and calcium were observed in females at 1000 mg/kg bw/day. The increase in A/G ratio noted in females at 300 mg/kg bw/day was not considered as an adverse effect because of no accompanying changes (see Table 2). Decreases in glucose and alkaline phosphatase observed in females of 100 mg/kg bw/day group and 300 mg/kg bw/day, respectively, were incidential observations and thus considered non-adverse.

NEUROBEHAVIOUR
No neurobehavioural abnormalities were observed in the test substance-treated groups.

ORGAN WEIGHTS
Increases in a relative weight of liver and absolute and relative weights of kidney for males and increases in relative weights of liver and kidney for females were observed in the 1000 mg/kg bw/day group (see Table 3).

GROSS PATHOLOGY
No adverse effects were observed for males and females.

HISTOPATHOLOGY: NON-NEOPLASTIC
Tendency of increase in hypertrophy of centrilobular hepatocytes and a reduction in the grade of periportal fatty change were observed in males of the 1000 mg/kg bw/day group (see Table 4).

Dose descriptor:

LOAEL

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: clinical chemistry; body weight; organ weight

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical chemistry; body weight; organ weight

Critical effects observed:

not specified

Table 1: Body weight gain in males

Days of treatment		dose [mg/kg bw/day]
		0	100	300	1000
males		13	13	13	13
1-7	mean	23.6	27.3	19.9	21.5
	SD	10.1	7.5	11.9	4.6
7-14	mean	27.4	26.1	23.4	27.3
	SD	7.9	6.8	6.1	5.6
14-21	mean	20.4	24.1	22.1	21.2
	SD	5.9	4	6.7	8.5
21-28	mean	24.4	25.2	24.1	20.8
	SD	8.8	4	7.9	5.4
28-35	mean	25.4	24.5	22.1	17.3**
	SD	5.3	5	4.3	5
35-42	mean	22.5	22.7	21	13.1**
	SD	6.2	4.7	5.4	6

Table 2: Altered clinical chemistry data

		dose [mg/kg bw/day]
		0	100	300	1000
males		13	13	13	13
A/G ratio	mean	1.18	1.16	1.21	1.28*
	SD	0.09	0.1	0.11	0.09
females		12	10	9	11
A/G ratio	mean	1.26	1.32	1.41*	1.45**
	SD	0.07	0.08	0.16	0.13
Creatinine [mg/dL]	mean	0.7	0.7	0.8	0.6**
	SD	0.1	0	0.1	0.1
T.protein [g/dL]	mean	5.4	5.4	5.5	5.1**
	SD	0.2	0.2	0.3	0.2
Ca [mg/dL]	mean	9.8	9.9	9.8	9.40**
	SD	0.2	0.3	0.4	0.3

 

Table 3: Selected organ weights

			dose [mg/kg bw/day]
			0	100	300	1000
males			13	13	13	13
liver	absolute [g]	mean	14.22	15.07	14.19	15.48
		SD	161.00	1.66	1.14	1.34
	relative [%]	mean	2.86	2.99	2.95	3.27**
		SD	0.16	0.26	0.17	0.21
kidney	absolute [g]	mean	3.23	3.23	3.31	3.64**
		SD	0.29	0.30	0.22	0.29
	relative [%]	mean	0.65	0.64	0.69	0.77**
		SD	0.06	0.07	0.04	0.06
females			12	10	10	11
liver	absolute [g]	mean	11.27	10.71	11.50	12.34
		SD	1.22	0.70	1.69	1.28
	relative [%]	mean	3.53	3.51	3.70	3.92**
		SD	0.31	0.22	0.27	0.24
kidney	absolute [g]	mean	2.09	1.99	2.20	2.28
		SD	0.18	0.21	0.19	0.22
	relative [%]	mean	0.65	0.66	0.72	0.72*
		SD	0.06	0.07	0.12	0.04

 

Table 4: Histopathological changes

			dose [mg/kg bw/day]
			0	100	300	1000
males			5	5	5	12
liver	hypertrophy, hepatocyte, centrilobular	very slight	0	0	0	5*
	Fatty change periportal	very slight	2	2	5	5
		slight	3	2	0	0
		moderate	0	1	0	0
	Total incidence		5/5	5/5	5/5	5/12**

 * significant difference from control; p>0.05

 ** significant difference from control; p>0.01

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

based on a structural analogue that is expected to represent the effects of the substance

System:

hepatobiliary

Organ:

liver

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

The study available is on the analogue bis(2-ethylhexyl) azelate. The effects of this analogue (C8) are considered to represent potential effects of diesters of alcohols, C7-9-iso-, C8-rich, 2-ethylhexyl and nonanedioic acid, as their toxicokinetic behaviour (including absorption) is expected to be very similar. Based on its starting materials this analogue is also expected to represent a worst case.

Justification for classification or non-classification

Based on the outcome of the available studies on the analogue substances, no classification for diesters of alcohols, C7-9-iso-, C8-rich, 2-ethylhexyl and nonanedioic acid is considered according to EC No 1272/2008.