Dehydrated sorbitol, C18 (unsaturated) fatty acid esters, ethoxylated

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

06 Aug - 07 Oct 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions. No data on analytical purity of test substance, only 2 dose levels, only organogenesis covered (days 6-15 of gestation).

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

no data on analytical purity of test substance, only 2 dose levels, only organogenesis covered (days 6-15 of gestation)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sorbitan monooleate, ethoxylated

EC Number:

500-019-9

EC Name:

Sorbitan monooleate, ethoxylated

Cas Number:

9005-65-6

Molecular formula:

Not applicable as UVCB

IUPAC Name:

Sorbitan, C18-unsat, fatty acid esters, ethoxylated (1 - 6.5 moles ethoxylated)

Constituent 2

Reference substance name:

Tween 80

IUPAC Name:

Tween 80

Details on test material:

- Name of test material (as cited in study report): Polyoxyethylene sorbitan monooleate (Polysorbate 80, Tween 80)
- Physical state: slightly amber, viscous liquid
- Analytical purity: no data
- Lot/batch No.: 120111
- Stability under test conditions: stable for 4 weeks when kept at room temperature protected from the light
- Storage condition of test material: refrigerator
- Composition of test material, percentage of components: a colorimetric assay indicated 86.3 ± 0.9% fatty acid esters and 13.7 ± 0.9% carbowaxes; the free fatty acid content was determined to be 1.06 ± 0.01% (0.9% RSD) expressed as oleic acid
- Other: identity of the test material was confirmed using infrared spectrometry

Test animals

Species:

rat

Strain:

other: Crl: CD ® BR VAF/PlusTM outbred albino rats

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA
- Age at study initiation: ca. 8-10 weeks (females), ca. 10-12 weeks (males)
- Weight at study initiation: 200-275 g (females) on gestational day (GD) 0
- Housing: sperm-positive females were individually housed in solid-bottom polycarbonate cages with stainless steel wire lids (Laboratory Products, Rochelle Park, NJ, USA ) and Ab-Sorb-Dri® cage litter (Laboratory Products, Garfield, NJ, USA)
- Diet: pelleted Purina Certified Rodent Chow® (#5002) (Ralston Purina Co., St. Louis, Missouri, USA), ad libitum
- Water: deionised/filtered water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25
- Air changes (per hr): 12-14
- Humidity (%): 50-81
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: deionised/distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: a predetermined amount of the test substance was weighed into volumetric flask or a tared, calibrated beaker. The appropriate amount of water was added in order to achieve the desired concentration. Formulations were shaken or stirred to achieve a visibly homogeneous mixture. Each concentration of the test substance was formulated independently once during the study in order to provide sufficient quantities for dosing.

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:

Prior to administration, each formulation was analysed to ensure a concentration between 90 and 110% of the target concentration by UV/VIS Spectrometry. The concentrations of the dose formulation of the test substance were analysed to be approx. 100% of the target concentration.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: after a 7-day acclimation period, individual breeding pairs were cohabited.
- M/F ratio per cage: 1/1
- Length of cohabitation: over night
- Further matings after two unsuccessful attempts: yes, sperm-negative females were returned to the male's cage and checked for sperm on successive mornings until insemination occurs or the treatment groups were filled, whichever came first.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy

Duration of treatment / exposure:

Day 6-15 of gestation

Frequency of treatment:

once daily, 7 days/week

Duration of test:

20 days

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The designated exposure levels were based upon the literature review as included in the study report.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once on the mornings of GD 0 through 20; more often than once per day, if necessary.

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were determined on the mornings of GD 0, 3, 6 through 15, 18 and 20, and immediately following sacrifice on GD 20.

FOOD CONSUMPTION: Yes, on GD 0, 3, 6, 9, 12, 15, 18 and 20.
- 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

WATER CONSUMPTION: Yes
- Time schedule for examinations: water consumption was determined on GD 0, 3, 6, 9, 12, 15, 18 and 20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: liver, heart and kidney (right) and uterus was weighed. Liver, heart and kidney (right) were stored in 10% neutral buffered formalin for optional histopathology.

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 resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

General Linear Models (GLM) procedures were applied for the analyses of variance (ANOVA) of maternal and foetal parameters (SAS® software). Prior to GLM analysis, an arcsine-square root transformation was performed on all litter-derived percentage data (Snedecor and Cochran, 1967) and Bartlett's test for homogeneity of variance was performed on all data to be analysed by ANOVA (Winer, 1962). GLM analysis determined the significance of dose-response relationships and the significance of dose effects. When ANOVA revealed a significant (p<0.05) dose effect, Williams' and Dunnett's Multiple Comparison Tests (Williams, 1971 and 1972; Dunnett, 1955 and 1964) compared treatment groups to control groups. One-tailed tests were used for all pairwise comparisons except maternal body and organ weights, maternal food and water consumption, foetal body weight and percent males per litter. Nominal scale measures were analysed by a χ2 test for independence and by a test for linear trend on proportions. When a χ2 test showed significant group differences, a one-tailed Fisher's exact probability test was used for pairwise comparisons of treated and control groups.
Except for nominal scale measures, data will be reported as mean value ± SEM (standard mean error).

REFERENCES: see under "Any other information on materials and methods incl. tables")

Historical control data:

Historical control data from teratologic studies in rats of the same strain investigated at the above mentioned laboratory were available.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: increased relative liver weight at both dose levels, non-adverse

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
No mortalities and treatment-related clinical signs of toxicity were observed for individual animals during the in-life phase of the study or at scheduled necropsy.

BODY WEIGHT AND WEIGHT GAIN
Average maternal body weight (on GD 0, 3, 6, 9, 12, 15, 18 and 20) did not differ among treatment groups, nor was there a treatment related change in maternal weight gain during treatment or gestation (absolute or corrected).

ORGAN WEIGHTS (see Table 1 under "Any other information on results incl. tables").
There were no treatment-related effects upon the following maternal organ weights: gravid uterine weight (absolute), kidney weight (absolute or relative) and heart weight (absolute or relative).
Relative maternal liver weight (% body weight on GD 20 or %corrected body weight) was statistically significantly elevated in both treatment groups, whereas absolute liver weight was only significantly increased at 500 mg/kg bw/day. However, due to the low increase in relative organ weight and the absence of any data on histopathological changes in liver, this effect was considered to be non-adverse.

FOOD AND WATER CONSUMPTION
Maternal food intake was comparable across groups during the pre- and posttreatment periods, but was decreased by -14% during the first 3 days of treatment at 5000 mg/kg bw/day relative to the vehicle control group.
Maternal relative water intake was comparable among treatment groups throughout gestation.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 5 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 5 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: increasing linear trend for percent foetuses with skeletal malformations per litter, non-adverse

Details on embryotoxic / teratogenic effects:
FETAL EXAMINATION
No adverse effects were noted on growth, viabilty or morphological development of fetuses. The overall incidence of malformations did not differ significantly among the treatment groups. Moreover, only a slight increase in the incidence of fetuses showing external, visceral or anatomical variations was determined in test animals after intrauterine exposure to 500 or 5000 mg/kg bw/day: However, except the skeletal anomalies, no dose-relationship was observed and hence, visceral and external effects are considered as incidental and not treatment-related. In the low dose group, one foetus (1/280 or 0.4%) exhibited any skeletal malformations (please refer to Table 2) visible as bipartite vertebra in the thoracic region (i.e. bipartite cartilage and bipartite ossification centres) which is a common finding in this species and strain. At 5000 mg/kg bw/day, 4 foetuses (4/341 or 1.4%) exhibited skeletal malformations: 3 foetuses exhibited only bipartite vertebra in the thoracic region (i.e. two with bipartite cartilage and bipartite ossification centres and one with bipartite cartilage and dumbbell ossification centre) and one foetus exhibited multiple skeletal malformations (agenesis of the lumbar, sacral and caudal vertebra; fused thoracic vertebra; fused ribs, ribs unattached to the sternum) accompanied by multiple external malformations (craniorhachischisis, short tail and umbilical hernia). However, as the fetal incidence of abnormal skeletal findings lay within the range of spontaneous skeletal anomalies determined on gestational day 20 in the same rat strain and comparable time frame (please refer to Table 3, MARTA, 1993), the determined skeletal abnormalities are not interpreted as adverse but rather spontaneous.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEC

Effect level:

>= 5 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: No effects

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1. Maternal liver weights (mean values ± SEM)

Absolute and relative weights	Dose (mg/kg bw/day)
	0	500	5000
Liver weight (g)	16.95 ± 0.35	18.48 ± 0.41 *	17.81 ± 0.35
% of body weight	4.24 ± 0.07	4.48 ± 0.07 *	4.47 ± 0.07 *
% corrected body weight	5.33 ± 0.09 §	5.65 ± 0.09 *	5.67 ± 0.08 *

* p<0.05: Dunnett's Test or Williams' Test

§ p<0.05: Test for Linear Trend

 

Table 2. Results of uterine and foetal examination

Uterine and foetal examinations			Dose (mg/kg bw/day)
			0	500	5000
All litters			20	19	23
No. Corpora lutea/dam^a			15.2 ± 0.7	15.6 ± 0.6	15.7 ± 0.5
No. Implantations/litter^a			14.8 ± 0.9	15.2 ± 0.6	15.5 ± 0.4
% Preimplantation loss/litter^a			4.4 ± 3.1	3.7 ± 1.0	2.7 ± 0.9
% Resorptions/litter^a			2.7 ± 1.4	2.9 ± 1.1	4.0 ± 1.4
% Litter with resorptions			25	32	35
% Late foetal deaths/litter^a			0.0 ± 0.0	0.0 ± 0.0	0.4 ± 0.4
% Litters with late foetal deaths			0	0	4
% Litter with 100% nonlive implants^b			0	0	0
% Adversely affected implants/litter^a,c			3.0 ± 1.4	6.2 ± 2.5	6.2 ± 1.5
% Litters with adversely affected implants^c			30	42	57
Live Litters^d			20	19	23
No. Live foetuses/litter^a			14.5 ± 0.9	14.7 ± 0.6	14.8 ± 0.5
% male foetuses/litter^a			52 ± 3	53 ± 3	47 ± 2
Mean foetal body weight (g)/litter^a		Both sexes	3.63 ± 0.06	3.71 ± 0.06	3.65 ± 0.05
		Male	3.72 ± 0.06	3.80 ± 0.07	3.74 ± 0.05
		Female	3.53 ± 0.06	3.59 ± 0.07	3.58 ± 0.05
% Foetuses with malformations/litter^a,e		external	0.0 ± 0.0	0.0 ± 0.0	0.3 ± 0.3
		visceral	0.3 ± 0.3	3.2 ± 2.1	0.6 ± 0.4
		skeletal	0.0 ± 0.0 §	0.3 ± 0.3	1.4 ± 0.6
% Litters with malformations^f		external	0	0	4
		visceral	5	21	9
		skeletal	0	5	17
% Fetuses with any malformation/litter^a,e		Both sexes	0.3 ± 0.3	3.5 ± 2.1	2.0 ± 0.7
		Male	0.5 ± 0.5	2.8 ± 2.3	1.8 ± 1.0
		Female	0.0 ± 0.0	3.5 ± 2.1	1.8 ± 1.0
% Litters with malformations^f			5	21	26
% Fetuses with variations/litter^a,e	Both sexes		17.9 ± 3.9	13.1 ± 2.9	19.8 ± 5.4
	Male		18.3 ± 3.6	13.6 ± 3.2	16.2 ± 4.9
	Female		18.1 ± 5.7	11.5 ± 3.4	22.6 ± 6.1
% Litters with variations^f			75	63	70

a Reported as the mean ± S.E.M.

b Nonlive = late foetal deaths plus resorptions

c Adversely affected = nonlive plus malformed

d Includes all dams with live foetuses; litter size = no. live foetuses

e Foetuses with one or more malformations or variations

f Litters with one or more foetuses with malformations or variations

§ p<0.05; Test for Linear Trend

 

Table 3.Skeletal Malformations in foetuses

	Dose (mg/kg bw/day)			Historical control data (taken from MARTA, 1993)
	0	500	5000
Total No. of foetuses examined^a	289	280	341
Total No. of litters examined^b	20	19	23
Skeletal malformations				Skeletal anomalies on GD 20
No. of foetuses with skeletal malformations^c	0	1	4
% Foetuses with skeletal malformation	0.0	0.4	1.2
No. of litters with skeletal malformations^d	0	1	4
% Litters with skeletal malformations	0.0	5.3	17.4
Fused ribs and fused rib cartilage			1 (0.29%)	0.10 – 0.68%
Rib cartilage not attached to sternum: ribs I-VII			1 (0.29%)	Branched/split: 0.03 – 0.4 Misshapen: 0.39 – 3.28
Bipartite cartilage. bipartite ossification centre: thoracic centrum		1 (0.36%)	2 (0.58%)	Misshapen: 0.07 – 0.66
Bipartite cartilage. dumbbell ossification centre: thoracic centrum			1 (0.29%)	Dumbbell-shaped: 2.14 – 21.48
Fused centra and fused arches: thoracic			1 (0.29%)	0.12 – 1.18
Agenesis of all vertebrae below lumbar II			1 (0.29%)	Lumbra: 0.16 – 1.84 Sacral: 0.04 – 0.48

a Only live foetuses were examined

b Includes only litters with live foetuses

c Foetuses with one or more malformations/variations

d Litters with one or more malformed/variant foetuses