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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

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).

Reproductive performance in males and females was not altered, but the oestrus cycle in females at 1000 mg/kg bw was slightly longer. No effects were found on fertility, copulation index and pregnancy rate. In females there was no dose related effect on the number of corpora lutea, implants, resorptions and gestation length. The number of (live) pups was lower (not reaching statistical significance) in litters from females treated at 1000 mg/kg bw. No effects on sex ratio or pup body weight were seen.

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

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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 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

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged: Individually

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

Details on study design:

- Dose selection rationale: The concentration was determined based on the result of pre-test.
The concentrations used in the pre-test were 250, 500 and 1000 mg/kg bw/day. There were no differences between the 250 and higher concentration treatment groups. This is the reason why the highest concentration used in this test was 250 mg/kg bw/day.

Parental animals: Observations and examinations:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at 13:00-16:00 before dosing and on Days 7, 14, 21, 28, 35 and 42 of the treatment period using scoring systems. In the detailed clinical observations, the animals were observed for about 20 min to determine changes in appearance, gait, posture, locomotor activity, grooming, respiratory rate, palpebral opening, and presence of vocalization, tail reaction, stereotype, tremor, convulsion, piloerection and abnormal behavior.

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 1 (before dosing), 7, 14, 21, 28, 35, 42 of treatment and prior to necropsy in males and on Days 1 (before dosing), 7 and 14, 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:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was not determined during the mating period in males and females.

OTHER:
Neurobehavioral tests, such as Preyer's reflex, pupillary reflex, pain reaction, withdrawal reflex, eyelid reflex and righting reflex, were determined for 5 animals/sex/dose on Day 42 of treatment in males and on day 4 of lactation in females.
Haematological and blood biochemical examination:
Blood samples were collected from the abdominal aorta after overnight starvation on next day of the last administration.

Oestrous cyclicity (parental animals):

Parameters examined in P female parental generation:
estrous cycle, precoital interval, frequency of vaginal estrus cycle and gestation length

Sperm parameters (parental animals):

Parameters examined in P male parental generation:
testis weight, epididymis weight, histopathology of male reproductive organs

Litter observations:

STANDARDISATION OF LITTERS
- Performed on Day 4 post partum: yes

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

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

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weight: Brain, heart, liver, kidney, spleen, adrenal, thymus, testis and epididymis
Microscopic examination: 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.

Postmortem examinations (offspring):

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

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

Statistics:

Dunnett's test for continuous data, and Mann-Whitney' U-test and Fisher' test for discrete data

Reproductive indices:

copulation index (no. of copulated pairs/no. of mated pairs x 100),
fertility index (no. of pregnant animals/no. of animals with successful copulation x 100),
gestation index (no. of females with live pups/no. of pregnant females x 100),
implantation index (no. of implantation sites/no. of corpora lutea x 100),
delivery index (no. of pups born/no. of implantation sited x 100)

Offspring viability indices:

birth index (no. of live pups on day 0/no. of implantation sites x 100),
live birth index (no. of live pups on Day 0/no. of pups born x 100),
viability index (no. of pups alive on Day 4 of lactation/no. of pups born x 100)

Clinical signs:

no effects 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):

effects observed, treatment-related

Description (incidence and severity):

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

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

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)

Other effects:

not examined

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: prolongation of estrous cycle

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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 FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight gain was suppressed in males of the 1000 mg/kg bw/day-treated group. No effects were observed in males and females of the test substance-treated groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
At 1000 mg/kg bw/day, the test substance altered the length of estrous cycle.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No effects were observed in sperm parameters in males.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No effects observed on reproductive performance in males and females given each dose.

ORGAN WEIGHTS (PARENTAL ANIMALS)
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 chapter for repeated dose toxicity).

GROSS PATHOLOGY (PARENTAL ANIMALS)
No adverse effects were observed for males and females.

HISTOPATHOLOGY (PARENTAL ANIMALS)
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 chapter for repeated dose toxicity).

OTHER FINDINGS (PARENTAL ANIMALS)
An increase in albumin/globulin 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 albumin/globulin ratio noted in females at 300 mg/kg bw/day was not considered as an adverse effect because of no accompanying changes (see chapter for repeated dose toxicity).

Dose descriptor:

LOAEL

Remarks:

reproduction

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: estrous cycle

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: estrous cycle

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: overall effects

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

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
No treatment related effect was observed on total No. of pups born, No. of males, No. of females, sex ratio, live pups, stillbirth and runt on post-natal Day (PND) 0 and total No. of live pups and sex ratio on PND 4. Viability index was unaffected. Survival of the pups from PND 0 - 4 remained unaffected in all treatment groups.

BODY WEIGHT (OFFSPRING)
No effect on group mean litter weight, total litter weight, male litter weight and female litter weight on PND 0 and PND 4.

GROSS PATHOLOGY (OFFSPRING)
No substance related gross external and internal findings were observed in any of the treated groups.

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Reproductive effects observed:

not specified

Table 1: Reproductive parameters

Dose(mg/kg bw/day)		0	100	300	1000
No.of female animals:		13	13	13	13
No.of animals which type of cycle was changed after the treatment:		3	5	5	8
Length of estrous cycle (days)
	mean	4.1	4.3	4.3	4.6*
	SD	0.5	0.3	0.4	0.5
No. of mated pairs:		13	13	13	13
No. of copulated pairs:		13	13	13	13
Copulation index (%)		100	100	100	100
No. of pregnant females		12	11	11	12
Fertility index (%)		92.3	84.6	84.6	92.3
Pairing day until copulation
	mean	3.4	3.0	2.5	1.8
	SD	3.3	3.5	1.1	1.2
Frequency of vaginal estrus during mating period
	mean	1.0	1.0	1.0	1.0
	SD	0.0	0.0	0.0	0.0
No. of pregnant females with pups alive:		12	11	11	12
Gestation index (%)		100.0	100.0	100.0	100.0
Gestation length (days)
	mean	22.6	22.4	22.7	22.9
	SD	0.5	0.5	0.5	0.3
No. of corpora lutea
	mean	16.2	16.4	16.6	17.4
	SD	1.3	1.7	1.9	2.9
No.of implantation site
	mean	15.5	15.8	16.1	14.9
	SD	1.2	1.7	1.7	4.0
Implantation index (%)
	mean	96.0	96.8	96.9	85.7
	SD	5.2	5.7	4.0	21.0
Day 0 of lactation
No.of pups born
	mean	13.8	14.9	14.5	12.3
	SD	2.0	2.7	1.9	5.1
Delivery index (%)
	mean	89.3	93.6	89.8	78.3
	SD	11.2	8.4	6.2	24.4
No.of pups alive
	mean	13.6	13.6	13.5	9.9
	SD	2.2	4.1	2.4	5.3
Birth index (%)
	mean	87.7	86.0	83.8	65.1
	SD	12.7	22.1	9.5	29.6
Live birth index (%)
	mean	98.1	92.4	93.5	84.1
	SD	4.8	23.0	10.6	28.0
Pups weight (g)
Male	mean	7.2	6.9	7.2	6.8
	SD	0.5	0.8	0.9	0.7
Female	mean	6.7	6.5	6.8	6.5
	SD	0.7	0.8	0.9	0.8
Sex ratio on day 0 of lactation (no. of male pups/total no. of pups)
	mean	54.1	47.0	45.9	48.5
	SD	11.5	12.0	17.6	24.5
Day 4 of lactation
No.of pups alive
	mean	13.1	14.2	13.7	9.6
	SD	3.1	2.7	1.9	5.2
Viability index (%)
	mean	95.6	97.7	98.1	97.8
	SD	13.1	7.4	5.9	3.8
Pups weight (g) on day 4 of lactation
Male	mean	11.6	11.0	11.7	10.9
	SD	1.0	2.3	2.5	2.4
Female	mean	10.9	10.5	11.3	11.0
	SD	1.5	2.2	2.4	0.7
Sex ratio on day 4 of lactation (no. of male pups/total no. of pups)
	mean	53.5	44.1	47.3	48.8
	SD	11.3	8.0	17.9	24.5

* significant difference from control, p < 0.05

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:

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

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

An effect on the oestrus cycle was observed at 1000 mg/kg bw, at which dose maternal toxicity becomes apparent.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

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.