Sorbitan tristearate

Toxicity to reproduction

Administrative data

Endpoint:

three-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given (analytical purity of test substance not given, 3-gen study, limited evaluation of parameters (e.g. sperm parameters, implantations, corpora lutea).

Data source

Reference

Reference Type:

publication

Title:

Three-generation reproduction study of rats ingesting up to 10 % sorbitol in the diet-and a brief review of the toxicological status of sorbitol

Author:

MacKenzie, K.M. et al.

Year:

1986

Bibliographic source:

Fd Chem Toxic 24/3: 191-200

Materials and methods

Principles of method if other than guideline:

A three-generation feeding study in rats was performed.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

D-glucitol

EC Number:

200-061-5

EC Name:

D-glucitol

Cas Number:

50-70-4

Molecular formula:

C6H14O6

IUPAC Name:

D-glucitol

Details on test material:

- Name of test material (as cited in study report): sorbitol 834 LF
- Physical state: crystalline
- Lot/batch No.: nos 5106 GO and 5050 Cl

Test animals

Species:

rat

Strain:

other: Charles River CD R (SD) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, USA
- Age at study initiation: P: 5 wks; F1/F2: at weaning
- Fasting period before study: not applicable
- Housing: singly during growth and holding phases in suspended wire-mesh cages with "deotised" animal cage boards as linings for the urine- and faeces-collecting pans; during mating, double-sized stainless-cages of similar design were used; pregnant rats and rats with young were housed in polycarbonate cages and provided with heat-treated hardwood chips
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (ad libitum): sucrose-containing (10%) ground cereal-based diet, obtained from Tekland Test Diets, Madison, USA
- Water (ad libitum): partially demineralised water
- Acclimation period: 2 weeks (P)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 30-70

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): The test substance was included in the basal diet at the expense of its sucrose content
- Storage temperature of food: after mixing, the diets were refrigerated for less than 48 hours (usually 24 hours) before being given to the animals; food was left in the food hoppers of cages for 7 days

Details on mating procedure:

- M/F ratio per cage: 1:2
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): singly

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of samples of each test diet taken at approximately 4-week intervals throughout the study showed that all were within 15% of the correct percentage. No details on method provided.

Duration of treatment / exposure:

P: 33 wks (14 wks before mating until they were killed in week 33)
F1: 38 wks (during lactation and then after weaning 14 wks until mating and subsequent until they were killed in week 68)
F2: 27 wks (during lactation and then after weaning 14 wks until mating and subsequent until they were killed in week 92)
F3: 3 wks (until weaning = day of sacrifice)

Frequency of treatment:

continously via diet

Details on study schedule:

- F1/F2 parental animals not mated until 19 weeks after being selected from the F1/F2 litters.
- Age at mating of the mated animals in the study: 14 weeks (P), 19 weeks (F1/F2)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
2.5, 5, 10%
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
2000, 4000, 8000 mg/kg bw/day
Basis:
other: actual ingested (based on an average daily food uptake of 20 g and and average body weight of 250 g)

No. of animals per sex per dose:

12 males, 24 females

Control animals:

yes, plain diet

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (pregnant and lactating females)
- Cage side observations checked: changes in appearance or activity, any indication of toxicity, including death

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during growth phase; females additionally on day 0, 7, 14 and 20 of gestation and on day 0, 4, 14 and 21 of lactation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes

OTHER:
Blood calcium was analysed in P and F2 adults in the control and high-level groups and in F1 animals of all groups, just before sacrifice using the Worthington calcium test procedure to see wether the known effect of sorbitol on calcium absorption was evident. The blood samples from the F3 control and high-dose animals were als analysed for T3, T4 and thyroid-stimulationg hormone.

Oestrous cyclicity (parental animals):

Vaginal smears were taken from each female daily during the mating period to ascertain the stage of the estrous cycle.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 / F3 offspring: on the day of delivery, the numbers of live and stillborn pups, individual live pup weights, sex ratios, and gross abormalities were recorded; after 4 days the number, sex and individual weights of pups were recorded; the numbers, sex and individual weights of pups were again recorded on day 14 and day 21 of lactation

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

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [when offspring was weaned]
- Maternal animals: All surviving animals [when offspring was weaned]

GROSS NECROPSY
- Gross necropsy of all animals (recording of body weight, examination of eyes, nose, mouth, skin and appendages; the cranium was removed and the brain, pituitary, and cranial nerves were examined in situ; the thoracic and abdominal cavities and their contents were examined grossly following an incision in the ventral body wall

HISTOPATHOLOGY / ORGAN WEIGHTS
Adrenals, brain, heart, kidneys, caecum, liver, pituitary, spleen, thymus and thyroid (with parathyroids) of the F3 generation were weighed. Gonads of all animals were weighed. Tissues from these organs (including three levels of the brain) and from the aorta, bone (tibia) with marrow, epididymides, oesophagus, eyes, colon, any lesions, lungs with mainstem bronchi, lymph nodes (cervical), pancreas, prostate, salivary gland (mandibular), seminal vesicles, skeletal muscle, small intestine (duodenum, ileum, jejunum), spinal cord (two levels), stomach, urinary bladder and uterus with cervix from all F3 rats were fixed in AFA fixative (80% ethanol, 40% formalin-glacial acetic acid, 90:10:5 by vol.), sectioned and read routinely. In addition, the sicatic nerve, skin with mammary gland, sternum and trachea from all F3 rats were fixed but not routinely sectioned.

Statistics:

Analysis of variance was used to examine differences in body weight, body-weight gain, feed consumption, organ weights and clinical chemistry data. Where significance was indicated, Dunnett´s test was performed to compare treatment with control means. The Kruskal-Wallis test was used to compare the number of pups born, the percentage born alive, percentage survival and sex ratios. Where significant differences were indicated, Dunn´s test was used to compare treatment with control means. Reproductive indices were analysed using contingency tables, for testing whether probability of successful mating or gestation was independent of the treatment group.

Reproductive indices:

Male fertility index: (no. of males shown to be fertile - as defined by a female giving birth to a litter / no. placed with females) x 100
Female fertility index: (no. of females producing a litter / no. mated) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

slightly reduced food consumption in F1 males and in body-weight gain in P, F1 and F2 animals

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

slightly reduced food consumption in F1 males and in body-weight gain in P, F1 and F2 animals

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Food consumption:
Control, P and F2: no effects
F1: reduced consumption in males and sporadic variations in females
Body weights:
Reduced body weight gain was observed in all generations in both sexes, with a more marked effect in females than in males and in the P than in the F1 and F2 generations (see table 1).
Body-weight gain of dams was unaffected during gestation, but significantly higher in the 10% dose group during lactation (see table 2).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No effects on fertility for male and females of all groups and generations were observed. This was based on the mean no. of days to mate and the mean length of gestation that was unaffected in all groups and generations. No more parameters indicative for fertility were given.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Beetween-group variations of gonadal weights were observed but considered to have no toxicological significance because they lacked consistency and were not accompained by any histologically-evident changes (see Table 3).

GROSS PATHOLOGY (PARENTAL ANIMALS)
Caecal enlargement was observed in all animals. Reduced spleen weights in high-dose females of the F1 and F3 were observed as well as higher mean weights of the ovaries in P a females and of the testes F2 males. Since effects were observed by chance and not observed in other generations and were not associated with mircroscopically detectable abnormalities, they were not assumed to be treatment-related (see table 3).

HISTOPATHOLOGY (PARENTAL ANIMALS)
No toxicologically significant or other changes attributable to treatment were detected.

OTHER FINDINGS (PARENTAL ANIMALS)
Serum calcium levels were elevated in P males and females exposed to 10% test substance and in F1 males exposed to 5 or 10% sorbitol. T3 and TSH levels in F3 animals were slightly differing from control animals without consistent pattern. No effects on haemoglobin was observed.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 10 other: % in diet (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Corresponding to 8000 mg/kg bw/day (based on an average daily food uptake of 20 g and and average body weight of 250 g)

2

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

>= 10 other: % in diet (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Corresponding to 8000 mg/kg bw/day (based on an average daily food uptake of 20 g and and average body weight of 250 g)

3

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 10 other: % in diet (nominal)

Based on:

test mat.

Basis for effect level:

other: Corresponding to 8000 mg/kg bw/day (based on an average daily food uptake of 20 g and and average body weight of 250 g of the parental animals)

Remarks on result:

other: Generation: F1, F2, F3 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

caecal enlargement in all animals

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

a lower incidence of hepatocytic swelling in high-dose group F3 females compared to controls was observed as well as higher mineral deposition in kidneys of control F3 females than in high-dose F3 animals (by chance finding)

Details on results (F1)

VIABILITY (OFFSPRING)
An apparently higher percentage of stillborn pups in the highest dose group animals of the F1 generation was counterbalanced by a higher level of stillborns in controls than in treated groups in the F2 and F3 generations. The same was true for comparisons of the percentages of pups that died between day 0 and 4 of lactation; more controls than treated F2 animals died during days 4-21 of lactation (see table 4).

BODY WEIGHT (OFFSPRING)
No treatment-related effects were observed. A significantly higher mean birth weight of F3 pups in the 5% dose group than in the control group was explained by the mean for the controls being spuriously low. No effects on body-weight gain was observed during days 0-4 of lactation. However, the 10% test substance diet was thereafter associated with slightly reduced mean weights gains in the F1 and F2 pups and the 5% diet was similarly associated with a reduction in weight gain during lactation in the F2 generations.

HISTOPATHOLOGY (OFFSPRING)
A lower incidence of hepatocytic swelling in high-dose group F3 females compared to controls was observed as well as higher mineral deposition in kidneys of control F3 females than in high-dose F3 animals. Both effects were assumed to be chance findings and not-treatment related.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Mean body weights in P, F1 and F2 generations fed 0 -10% test substance in diet

		Males				Females
Time from weaning (wk)	Dietary level (10%	0	2.5	5	10	0	2.5	5	10
	P generation
0		165	158	160	158	135	128**	131	130
4		365	362	350	353	229	218	221	211*
8		448	455	431	439	272	256*	263	252**
15		537	538	511	513	315	296*	303	286**
0-15	Weight gain	372	380	351	355	180	168	172	153
	F1 generation
0		170	161	170	157	142	137	139	131
4		386	382	384	349	238	235	225	229
8		494	492	478	443*	284	280	269	268
15		573	574	557	517	318	315	296*	296*
0-15	Weight gain	403	413	387	360	176	178	157	165
	F2 generation
0		162	170	191	180	136	140	150	138
4		376	387	400	371	229	237	227	230
8		494	503	502	477	277	286	272	272
15		568	578	574	539*#	305	321	304	302
0-15	Weight gain	406	408	383	359	171	181	154	164

#: difference was significant at wk 13 but not at wk 14

*p < 0.05, **p < 0.01 (means for groups of 12 males and 24 females)

Table 2: Mean body weights during gestation and lactation for three generations of dams fed 0 -10% test substance in diet

			Mean body weight on gestation day (g):
Generation	Dietary level (%)	No. of dams	0	7	14	20	Weight change (g) on days 0-20
P dams/F1 offspring	0	18	335	361	387	455	+120
	2.5	20	321	345	372	438	+116
	5	17	323	348	374	441	+119
	10	22	315	334	358	432	+116
F1 dams/F2 offspring	0	15	348	376	404	464	+116
	2.5	17	359	389	420	497*	+138*
	5	16	330	359	392	463	+133
	10	19	328	350*	379	453	+126
F2 dams/F3 offspring	0	15	308	339	367	432	+125
	2.5	20	318	347	375	442	+125
	5	20	301	333	362	427	+126
	10	20	295	321	346	413	+118
		Mean body weight on lactation day (g):
Generation	Dietary level (%)	No. of dams #	0	4	14	21	Weight change (g) on days 0-21
P dams/F1 offspring	0	20	372	376	378	356	-15
	2.5	20	359	370	364	341	-17
	5	18	356	362	370	347	-9
	10	22	350	361	375	361	+13**
F1 dams/F2 offspring	0	17	383	388	389	371	-12
	2.5	18	398	413	410	380	-18
	5	18	378	390	396	370	-8
	10	21	357	378	399	368	+11**
F2 dams/F3 offspring	0	16	347	348	356	339	-8
	2.5	21	356	359	367	345	-12
	5	21	347	349	361	334	-14
	10	20	323	337	347	345	+22**

#: The numbers of dams weighed during lactation exceeded, in some groups, the number weigehd during gestation because no vaginal plugs or sperm was seen in some animals that subsequently delivered litters; as these rats were not identified as "pregnant", their gestation body-weight data were not recorded, but they were weighed druing lactation.

*p < 0.05, **p < 0.01

Table 3: Effect of multigeneration feeding of 0 -10% dietary test susbtance on the weights of gonads, spleen and caecum

		Mean organ weights (g) and (in brackets) relative organ weight (g/100 g bw):
Sex	Dietary level (%)	P adults	F1 weanlings	F1 adults	F2 weanlings	F2 adults	F3 weanlings
Gonads
M	0	3.63 (0.58)	0.27 (0.5)	3.81 (0.59)	0.3 (0.51)	3.65 (0.6)	2.99 (0.91)
	2.5	3.62 (0.58)	0.26 (0.5)	3.92 (0.59)	0.29 (0.51)	3.69 (0.61)	2.82 (0.86)
	5	3.53 (0.58)	0.23 (0.48)	3.75 (0.59)	0.28 (0.52)	3.68 (0.62)	2.91 (0.9)
	10	2.45 (0.58)	0.26 (0.54)	4.02 (0.66)	0.28 (0.52)	4.05 (0.71)**	3.16 (0.97)
F	0	0.12 (0.034)	0.03 (0.063)	0.13 (0.034)	0.04 (0.071)	0.13 (0.039)	0.1 (0.049)
	2.5	0.12 (0.035)	0.04 (0.09)	0.14 (0.035)	0.04 (0.078)	0.15 (0.043)	0.1 (0.047)
	5	0.12 (0.035)	0.03 (0.071)	0.12 (0.032)	0.04 (0.08)	0.15 (0.044)	0.1 (0.045)
	10	0.15** (0.044)**	0.03 (0.068)	0.13 (0.037)	0.03 (0.064)	0.15 (0.046)	0.1 (0.046)
Spleen
M	0		0.27 (0.49)		0.32 (0.56)		0.79 (0.24)
	2.5		0.24 (0.45)		0.32 (0.56)		0.85 (0.26)
	5		0.23 (0.47)		0.32 (0.59)		0.82 (0.25)
	10		0.22 (0.46)		0.3 (0.57)		0.78 (0.24)
F	0		0.26 (0.5)		0.31 (0.61)		0.54 (0.26)
	2.5		0.22* (0.44)		0.3 (0.56)		0.57 (0.26)
	5		0.24 (0.5)		0.3 (0.59)		0.64* (0.3)
	10		0.2** (0.44)		0.29 (0.56)		0.64* (0.3)
Caecum
M	0		0.68 (1.23)		0.3 (0.53)		1.47 (0.45)
	2.5		0.68 (1.32)		0.3 (0.54)		1.72* (0.53)
	5		0.76* (1.53)		0.33 (0.62)*		1.82** (0.57)**
	10		0.73* (1.49)		0.31 (0.58)		2.36** (0.72)**
F	0		0.61 (1.16)		0.27 (0.52)		1.09 (0.53)
	2.5		0.62 (1.26)		0.28 (0.52)		1.23 (0.57)
	5		0.67 (1.41)		0.32* (0.61)*		1.32* (0.61)
	10		0.73 (1.56)**		0.29 (0.58)		1.81** (0.85)**

Table 4: Effects of feeding 0 -10% dietary test substance on various pup indices

Parameter	Dietary level (%)	Generation
		F1a	F1b	F2a	F2b	F3a
Mean no. of pups/litter	0	13.2	12.4	11.3	10.8	13.6
	2.5	12.4	12.2	13.6	13.8**	12.9
	5	10.4	12.1	12.8	13.1*	11.1
	10	12	13	13.1	12.8	13
Pups stillborn (% of total)	0	1.5	2.2	3.3	2.4	5
	2.5	2.5	0.7	0.7	1.6	1.6
	5	3.2	2.4	0.8	1.1	0.7
	10	8	5.3	1.6	0	2
Pups deaths on days 0-4 of lactation (%)	0	1.9	9.2	15.5	7.4	3.1
	2.5	4.9	7.3	1.9	1.3	2.4
	5	4.2	3.5	3.2	2.7	2.4
	10	16.6#	5.8	2.8	1.7	0.9
Pups deaths on days 4-21 of lactation (%)	0	0	4	7.7	6.8	0
	2.5	2.4	0.7	0	0	0
	5	2.6	2.2	0.6	2.1	0
	10	3.5	2.5	3.3	2.4	0.6
Mean pup weight (g) on day 0	0	6.7	6.6	6.4	6.8	6
	2.5	6.7	6.7	6.6	6.8	6.4
	5	6.8	6.6	6.5	6.6	6.8**
	10	6.4	6.6	6.6	6.7	6.5

#: The standard deviation for this value was approximately 6, and the difference between 1.9% deaths for the controls and 16.6% deaths for the high-level exposure group was not statistically significant

*p < 0.05, **p < 0.01 (means for 15 -22 litters)

Applicant's summary and conclusion

Conclusions:

The test substance had no effect on reproductive performance.