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

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication which meets basic scientific principles

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1992

Materials and methods

Principles of method if other than guideline:

Lifespan (2-year) repeated dose toxicity study in mice at dietary concentrations of 25000 and 50000 ppm

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): Polysorbate 80
- Physical state: translucent, pale yellow, viscous liquid
- Analytical purity: approx. 83- 85%
- Composition of test material, percentage of components: approx. 83-85% sorbitan polyethylene glycol fatty acid esters and approx. 15-17% sorbitan polyethylene glycols
- Lot/batch No.: 250-1 and 7230-C
- Storage condition of test material: protected from light

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Frederick Cancer Research Facility (Frederick, MD, USA)
- Age at study initiation: 57 days (males), 64 days (females)
- Weight at study initiation: group mean (males): 22.2-22.7 g, group mean (females): 16.4-16.8 g
- Housing: animals were housed in groups of five in solid-bottom polycarbonate cages (Lab Products, Inc., Garfield, NJ, USA), equipped with BetaChip® hardwood laboratory bedding (Northeastern Products Corp., Warrensburg, NY, USA), reemay spun-bonded polyester cage filters (Snow Filtration, Cincinnati, OH, USA) and stainless steel racks (Lab Products, Inc., Garfield, NY, USA).
- Diet: NIH-07 Open-Formula Mash (Zeigler Bros.,Inc., Gardners, PA, USA), ad libitum
- Water: tap water via automatic watering system (Edstrom Industries, Inc., Waterford, WI, USA), ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8-33.3
- Humidity (%): 18-82
- Air changes (per hr): min. 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 Aug 1982 To: 07 Aug 1984

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): the dose formulation were prepared weekly during the study period.
- Mixing appropriate amounts with (Type of food): NIH-07 Open-Formula Mash
- Storage temperature of food: at 5 °C in the dark

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability analyses of the dosed feed preparations were analysed using UV spectroscopy. Therefore, samples were extracted with solvent, evaporated to dryness, resuspended in methylene chloride and reacted with cobalt thiocyanate solution. The absorbance of the methylene chloride layer was determined at 320 nm using UV spectroscopy. Homogeneity of these formulations was confirmed, stability of the formulation was established for at least 2 weeks when stored in the dark at temperatures up to 5 °C. During the 2-year studies, the dose formulations were analysed at least once every 8 weeks. Concentration analysis of the dose formulations showed that 97% (84/87) dose formulations were within the specified ± 10% of the target concentrations.

Duration of treatment / exposure:

103 weeks
interim sacrifice after 15 months

Frequency of treatment:

daily (7 days/week)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
25000 and 50000 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
5003 and 10811 mg/kg bw/day (male)
Basis:
other: based on the average reported body weight and food consumption (see Table 1 under “Any other information on results incl. tables")

Doses / concentrations 3

Remarks:

Doses / Concentrations:
6487 and 13197 mg/kg bw/day (female)
Basis:
other: based on the average reported body weight and food consumption (see Table 2 under “Any other information on results incl. tables")

No. of animals per sex per dose:

50 (main study control and treatment groups)
7-10 (interim sacrifice control and treatment groups)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: the dose levels were selected based on a previous 13-week toxicity study in mice, showing no adverse effects in mice up to the maximum dose of 50000 ppm. Thus, 50000 ppm was selected as the high dose and 25000 ppm was selected as the low dose for male and female mice in the 2-year study. Doses greater than 50000 ppm were not considered appropriate for the 2-year study, because diets containing more than 50000 ppm of the test substance could lead to nutritional deficiencies in the study animals.
- Rationale for animal assignment (if not random): animals were grouped by body weight and assigned to cages. Cages were assigned to treatment groups using a table of random numbers.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed twice daily and at study termination and clinical observations were recorded once per week, then monthly or as necessary thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed at study initiation, weekly for 13 weeks, and monthly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes, monthly
- 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: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, complete necropsy (all tissues and organs) was performed on all animals. Absolute and relative organ weights of brain, right kidney, and liver of all animals evaluated at 15 months (7-10 animals per each group) were determined at necropsy.
HISTOPATHOLOGY: Yes, complete histopathology was performed on all animals that died or were killed moribund prior to study termination, all animals from the 15-month interim evaluation that received 0 or 50000 ppm and all mice from the 0 and 50000 ppm dose groups that survived to study termination. Tissues examined included: adrenal gland, bone and bone marrow, brain (frontal cortex and basal ganglia, parietal cortex and thalamus, and cerebellum and pons), oesophagus, gallbladder, gross lesions and tissue masses, heart, kidney, large intestine (cecum, colon, and rectum), liver, lungs and mainstem bronchi, mammary gland, mandibular or mesenteric lymph nodes, nasal cavity and turbinates, ovary, pancreas, parathyroid gland, pituitary gland, prostate gland, salivary gland; seminal vesicle, skin, small intestine (duodenum, jejunum, and ileum), spleen, stomach, testis (with epididymis), thymus, thyroid gland, trachea, urinary bladder, and uterus.

Statistics:

Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone's (1975) life table test to identify dose-related trends. Ball reported p values for the survival analyses were two sided. Organ and body weight data, which have approximately normal distributions, were analysed using the multiple comparison procedures of Williams (1971 and 1972) and Dunnett (1955). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of dose-response trends and to determine whether a trend-sensitive test (Williams’ test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-response (Dunnett’s test).

REFERENCES:
Cox, D.R. (1972). Regression models and life tables. J. R. Stat. Soc. B34: 187-220.
Tarone, R.E. (1975). Tests for trend in life table analysis. Biometrika 62: 679-682.
Williams, D.A (1971). A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics 27: 103-117.
Williams, D.A. (1972). The comparison of several dose levels with a zero dose control. Biometrics 28: 519-531.
Dunnett, W. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50: 1095-1121.
Jonckheere, A. (1954). A distribution-free k-sample against ordered alternatives. Biometrika 41: 133-145

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

50000 ppm (females): reduction (-11% compared to controls) in final mean body weight

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Control group and 25000 ppm: increased incidence of squamous hyperplasia and inflammation of the forestomach (m/f); 50000 ppm: stat. signif. increase in the incidence of inflammation and squamous hyperplasia of the forestomach (m/f) as well as ulcers (f)

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Survival of male and female mice of both treatment groups was similar to that of the controls. Three control males, three males of the low dose group (25000 ppm) and one control female died prior to the 15-moth interim evaluations. As mortalities occurred only within the low dose group and also in the control group, they were not considered to be associated with treatment of the test substance.
No clinical findings associated with treatment of the test substance were observed in animals of both dose groups.

BODY WEIGHT AND WEIGHT GAIN
A slight reduction (-11%) in final mean body weights of females from the high dose group was observed compared to controls.
In contrast, no alterations in mean body weights and body weight gain were observed in treated males of both dose groups as well as treated females from the low dose group when compared to controls.

FOOD CONSUMPTION AND COMPOUND INTAKE
The average feed consumption of treated animals from both dose groups was similar to that of the controls. The average compound intake (determined over 101 weeks) for males and females of both dose groups is given in Table 1 and 2 under “Any other information on results incl. tables”.

ORGAN WEIGHTS
No changes in absolute and relative organ weights of brain, right kidney, and liver of all animals (7-10 per group) evaluated at 15 months were observed compared to the respective control group.

GROSS PATHOLOGY
No treatment-related gross pathological findings were observed in treated animals at the 15-month interim sacrifice and at the end of the 2-year study period.

HISTOPATHOLOGY: NON-NEOPLASTIC
During the 15-month interim evaluations, compound-related lesions occurred in 5 of 10 females from the high dose group. These lesions included mild hyperplasia of the forestomach epithelium and inflammation of the forestomach. No test substance-related non-neoplastic lesions were observed in treated males of both dose groups.
At the end of the 2-year study period, compound-related lesions of the forestomach occurred in mice of all dose groups (including controls) (see Table 3 under “Any other information on results incl. tables”). These included squamous hyperplasia of the epithelium, inflammation, and ulcers. The incidences and severity of hyperplasia of the forestomach epithelium (hyperplasia, squamous) and inflammation of the forestomach were significantly increased in males and females that received the highest concentration in the diet (50000 ppm). Hyperplasia of the forestomach was characterized by focal, broad-based lesions usually located near the limiting ridge between the forestomach and the glandular stomach. In the low dose group, minimal to mild hyperplasia consisted of slight thickening and folding of the epithelium; in marked lesions the thickened epithelium sometimes formed multiple projections into the lumen. Some hyperplasias were focal, elevated thickenings surrounding an ulcer. Hyperplasias were typically more extensive in high-dose than in low-dose animals. In a number of mice, aggregates of small to moderate numbers of neutrophils (inflammation, acute) were present in the forestomach wall. In several animals of the high dose group, chronic active inflammation, a scattering of small to moderate number of mixed Iymphocytes, macrophages, and neutrophils, often accompanied by varying amounts of fibrosis, were present in the forestomach wall beneath areas of hyperplasia. The forestomach squamous hyperplasia and inflammation in high-dose animals and the ulcers in high-dose females were considered to be related to treatment with the test substance.

HISTOPATHOLOGY: NEOPLASTIC
During the 15-month interim evaluations and at the end of the 2-year study period, no statistically significantly increased incidences of neoplasms occurred in treated mice of either sex. Although hyperplasia and inflammation was increased in animals of the high dose group, no neoplasms in the forestomach of these animals were noted. The only observed forestomach neoplasm, which were not considered to be of toxicological relevance, involved one squamous papilloma in a control male and one squamous cell carcinoma in a low-dose male.

Effect levels

1

Dose descriptor:

LOAEL

Effect level:

50 000 ppm

Based on:

test mat.

Remarks:

corresponding to 10811 (m) and 13197 (f) mg/kg bw/day based on the average reported body weight and food consumption (over 102 weeks)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Effect level:

25 000 ppm

Based on:

test mat.

Remarks:

corresponding to 5003 (m) and 6487 (f) mg/kg bw/day based on the average reported body weight and food consumption (over 102 weeks)

Sex:

male/female

Basis for effect level:

other: slight, but not significant increase in the incidence of squamous hyperplasia (minimal to mild) and inflammation of the forestomach

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1. Calculation of doses in mg/kg bw/day (males) over the 2-year study period from the reported food and compound consumption

Week	25000 ppm			50000 ppm
	Feed (g/day)	Body weight (g)	Dose (mg/kg bw/day)	Feed (g/day)	Body weight (g)	Dose (mg/kg bw/day)
1	7.5	22.7	8207	8.0	22.4	17860
2	6.0	25.6	5824	6.6	25.5	12889
4	4.7	27.5	4281	6.1	26.5	11481
5	7.6	28.0	6769	7.5	27.5	13641
9	8.3	30.5	6839	8.1	30.3	13316
13	8.5	31.8	6691	8.6	31.6	13553
16	10.2	33.5	7649	10.1	32.9	15365
20	9.7	34.6	7001	8.6	34.4	12513
25	8.8	35.7	6175	9.5	34.9	13637
29	9.5	35.5	6688	9.8	35.1	13984
33	9.4	36.8	6414	10.0	35.8	13952
37	8.2	35.4	5807	9.3	35.8	13039
41	10.2	37.4	6803	11.6	36.3	15936
45	11.3	36.8	7690	13.0	36.7	17724
49	10.6	38.4	6919	12.4	38.4	16166
53	9.3	38.3	6093	10.3	37.7	13637
58	4.3	37.4	2903	4.1	37.5	5509
62	4.5	36.2	3113	5.0	37.5	6614
66	4.7	38.4	3031	5.2	38.3	6851
70	5.0	36.8	3385	5.0	37.2	6718
74	4.3	38.5	2817	4.8	38.2	6339
78	4.0	38.3	2615	4.4	37.7	5859
82	4.3	37.5	2842	4.9	37.0	6564
86	3.9	36.1	2731	4.5	36.4	6119
90	3.8	31.7	2527	4.0	36.9	5364
94	4.3	36.4	2956	4.3	36.9	5761
98	3.8	36.5	2623	4.4	37.6	5915
102	3.9	36.2	2703	4.5	35.0	6399
Mean values over 102 weeks	6.8	34.6	5003	7.3	34.6	10811

Table 2. Calculation of doses in mg/kg bw/day (females) over the 2-year study period from the reported food and compound consumption

Week	25000 ppm			50000 ppm
	Feed (g/day)	Body weight (g)	Dose (mg/kg bw/day)	Feed (g/day)	Body weight (g)	Dose (mg/kg bw/day)
1	7.6	16.4	11624	7.9	16.5	24050
2	7.0	18.7	9354	6.5	18.9	17159
4	5.6	20.3	6838	5.1	20.3	12529
5	7.2	20.6	8787	7.1	20.3	17417
9	9.2	23.0	9980	8.8	22.8	19403
13	8.4	22.8	9208	9.3	22.8	20308
16	10.7	24.2	11094	10.7	24.7	21642
20	10.2	26.1	9816	9.7	25.3	19224
25	9.0	27.5	8173	10.2	26.6	19106
29	9.5	28.0	8498	8.4	27.7	15104
33	10.3	29.3	8763	10.0	28.1	17710
37	7.9	29.2	6772	7.8	28.2	13749
41	9.5	30.9	7696	10.0	29.4	17019
45	12.8	30.7	10429	11.0	28.8	19157
49	9.7	32.1	7543	9.7	30.7	15873
53	9.0	33.0	6835	7.6	30.8	12322
58	3.8	32.9	2864	3.5	31.3	5600
62	5.4	35.0	3880	5.8	32.9	8797
66	5.4	35.8	3746	5.8	33.1	8791
70	5.9	36.1	4076	5.4	33.5	8003
74	5.1	36.0	3542	5.0	34.2	7338
78	4.5	37.0	3045	4.8	34.4	6945
82	4.8	37.7	3211	4.9	34.4	7176
86	5.0	37.0	3407	6.0	34.7	8655
90	3.4	36.6	2337	3.7	34.4	5375
94	5.1	37.0	3439	5.3	35.1	7524
98	5.5	37.2	3689	4.9	34.7	7125
102	4.6	38.1	2993	4.6	35.6	6404
Mean values over 102 weeks	7.2	30.3	6487	7.1	28.9	13197

Table 3. Incidences of selected forestomach lesions in mice in the 2-year feeding study

	Control (0 ppm)	25000 ppm	50000 ppm
Male
Hyperplasia, squamous	3/48 (6%)	4/50 (8%)	19/50 (38%)**
Inflammation, acute	0/48 (0%)	3/50 (6%)	5/50 (10%)*
Inflammation, chronic active	0/48 (0%)	1/50 (2%)	7/50 (14%)**
Female
Hyperplasia, squamous	4/49 (8%)	8/50 (16%)	26/49 (53%)**
Inflammation, acute	2/49 (4%)	4/50 (8%)	3/49 (6%)
Inflammation, chronic active	2/49 (4%)	0/50 (0%)	13/49 (27%)**
Ulcer	1/49 (2%)	0/50 (0%)	7/49 (14%)*
* Significantly different (P<0.05) from the control group by the logistic regression tests ** P<0.01