Sucrose and glycerol, reaction products with C12-18, C18unsatd. fatty acids

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: combined repeated dose and reproduction / developmental screening

Type of information:

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

Adequacy of study:

key study

Study period:

2008-09-25 to 2008-11-14

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study performed on a supporting substance (structural analogue). In accordance with the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

March 1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Isostearic acid, esters with methyl α-D-glucoside

IUPAC Name:

Isostearic acid, esters with methyl α-D-glucoside

Constituent 2

Reference substance name:

138985-20-3

Cas Number:

138985-20-3

IUPAC Name:

138985-20-3

Test material form:

semi-solid (amorphous): gel

Remarks:

migrated information: paste

Test animals

Species:

rat

Strain:

other: Wistar Han (Crl:WI(Han))

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation (F0-treatment): approximately 10 weeks
- Weight at study initiation: male:279 - 317 g, female: 180 - 215 g
- Fasting period before study: no
- Housing:
Pre-mating: animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIII type, height 18 cm)
Mating: females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/sex/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Offspring was kept with the dam until termination in Macrolon cages (MIII type, height 18 cm)
- Diet: ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, tap-water
- Acclimation period F0: at least 5 days prior to start of treatment


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C (actual range: 18.4 to 22.2°C)
- Humidity (%): 30 - 70% (actual range: 37 - 94%)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% aqueous carboxymethyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Dose volume: 5 mL/kg bw. Actual dose volumes were calculated according to the latest body weight.

VEHICLE
- 1% aqueous carboxymethyl cellulose

TEST SUBSTANCE FORMULATION
- Stability of test substance in vehicle: stable in 1% aqueous carboxymethyl cellulose for at least 6 hours at room temperature over the concentration range 10 to 200 mg/mL (determined during this project).
- Method of formulation: formulations (w/w) were prepared daily, were homogenised to a visually acceptable level and dosed as soon as possible after preparation with a maximum of 2.5 hours after preparation. No adjustment was made for specific gravity of the test substance, vehicle, and/or
formulation. In order to obtain homogeneity, the test substance formulations were heated in a water bath with a maximum temperature of 45 °C for a maximum of 22 minutes. The test substance formulations were allowed to cool down to a temperature at a maximum of 40°C prior to dosing.
Based on results of the thermal analysis performed by NOTOX (NOTOX project 488541), reaction and/or decomposition of the test substance were observed above approximately 175°C and, therefore, the test substance was considered to be stable at 45°C.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: a minimum of 14 days of exposure
- Proof of pregnancy: sperm in vaginal lavage or by appearance of an intravaginal copulatory plug referred to as day 0 post coitum
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon cages (MIII type, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analyses were performed on a single occasion after the treatment phase according to a validated method (NOTOX Project 488541).
- The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115%. Homogeneity was demonstrated if the coefficient of variation was <= 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

Offspring: not treated
Males: exposed for 30 days, i.e, 2 weeks prior to mating, during mating, and up to termination
Females: exposed for 42-44 days, i.e, during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation

Frequency of treatment:

- Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Details on study schedule:

- Parturition F0:
The females were allowed to Iitter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.
- Lactation F0:
Examination of maternal care revealed no deficiencies (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding).

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 150 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

This study is described in full detail under chapter 7.5.1 "Repeated dose toxicity: oral".

Positive control:

no positive control group

Examinations

Parental animals: Observations and examinations:

This study is described in full detail under chapter 7.5.1 "Repeated dose toxicity: oral".

Oestrous cyclicity (parental animals):

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- Corpora lutea

Sperm parameters (parental animals):

All recorded microscopic findings were within the range of background pathology encountered in Wistar rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats. The spermatogenic staging profiles were normal for all group 1 and group 4 males evaluated.
Parameters examined in [all/P/F1/F2] male parental generations:
testis weight, epididymis weight, prostate weight, seminal vesicles, spermatogenesis staging

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in offspring:
- Number and sex of pups: on day 1 and 4 of lactation (by assessment of the ano-genital distance)
- Stillbirths, live births, postnatal mortality, if possible defects or cause of death: at day 1 of lactation and daily thereafter
- Clinical signs (detailed clinical observations, including abnormal behaviour): at least once daily
- Body weights: live pups were weighed during lactation on Days 1 and 4.

PATHOLOGY OFFSPRING
- Pups were killed by decapitation on Day 5 of lactation or shortly thereafter.
- The stomach was examined For the presence of milk.
- Descriptions of all external abnormalities were recorded. If possible, detects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin, for possible further examination.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
- Dose groups: the first 5 mated males per group and the first 5 females with live offspring per group
-- Macroscopic examination: Adrenal glands, Aorta, Brain (cerebellum, mid-brain, cortex), Caecum, Cervix, Clitoral gland, Colon, Coagulation gland, Duodenum, Epididymides,
(Eyes with optic nerve (if detectable) and Harderian gland), (Female mammary gland area), (Femur including joint), Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung, Ovaries, Pancreas, Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles, (Skeletal muscle), (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid includinq parathyroid (if detectable), (Tongue), Trachea, Urinary bladder, Uterus, Lymph nodes - mandibular, mesenteric, (Nasopharynx), Oesophagus, Vagina, all gross lesions

- From all remaining animals:
Cervix, Clitoral gland, Coagulation gland, Epididymides,Ovaries, Preputial gland, Prostate gland, Seminal vesicles, Testes, Uterus, Vagina, all gross lesions

- Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

-- Organ weights:
- From the first 5 mated males per group and the first 5 females with live offspring per group: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Prostate (when fixed for at least 24 hours), Seminal vesicles, Spleen, Testes, Thymus

- From all remaining males:
Epididymides, Testes, Prostate (when fixed for at least 24 hours), Seminal vesicles


HISTOPATHOLOGY: Yes
- Histopathologic examination was performed on an extensive list of organs and tissues from five males and five females of groups 1 and 4 as well as gross lesions from all rats. Sections of testes from five group 1 and 4 rats were assessed for spermatogenesis staging.
- Adrenal glands, aorta, bone - sternum [and femur including joint]; bone marrow - sternal, brain, clitoral glands, epididymides, esophagus, [eyes with optic nerve and Harderian glands); heart, [identification marks], kidneys, [Iacrimal glands - exorbital], large intestine cecum, colon and rectum; [larynx), liver, lungs, Iymph nodes - mandibular and mesenteric; [female mammary gland area], [nasopharynx], ovaries, pancreas, pituitary gland, preputial, glands, prostate gland, [salivary glands - mandibular and sublingual]; sciatic nerve, seminal vesicles with coagulation glands, [skeletal muscle], [skin], small intestine - duodenum, jejunum and ileum with Peyer's patches: spinal cord - cervical, midthoracic and lumbar; spleen, stomach, testes, thymus, thyroid glands with parathyroid glands, [tongue], trachea, urinary bladder, uterus with uterine cervix, vagina and all organs or tissues with macroscopic abnormalities.
Following fixation, organs (except those listed in brackets) from the selected animals of groups 1 and 4 along with all organs or tissues with macroscopic abnormalities from all rats, were trimmed , processed and embedded in paraffin wax, precision cut and stained with hematoxylin and eosin.

Postmortem examinations (offspring):

Pups were killed by decapitation on Day 5 of lactation or shortly thereafter.

All offspring was sexed and externally examined. The stomach was examined for the presence of milk. Descriptions of all external abnormalities were recorded. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin, for possible further examination.
No treatment-related changes were noted for reproduction, breeding and pup development.

Statistics:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data.
- No statistical analysis was performed on histopathology findings
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- Corpora lutea

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- Staging of Spermatogenesis

For further details please find under chapter: 7.5.1 "Repeated dose toxicity: oral".

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

3

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: - No treatment-related changes on reproduction, breeding and pup development).

Remarks on result:

other: Generation: reproduction, breeding, development (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:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
- Viability index (=Number of alive pups before planned necropsy/Number of pups born alive): 0 mg/kg bw/d: : 100% , 50 mg/kg bw/d: : 100%, 150 mg/kg bw/d: : 99.2%, 1000 mg/kg bw/d: : 98.4% (significant at 5 %)
- Dead pups at first litter check: 0 mg/kg bw/d: 3 males, 2 females; 50 mg/kg bw/d: 1 male, 1 female, 150 mg/kg bw/d: 0; 1000 mg/kg bw/d: 1 female
- Dead pups post natal: at 0, 50 mg/kg bw/d: 0, at 150 mg/kg bw/d: 1 male, at 1000 mg/kg bw/d: 1 male, 1 female from one litter
- 10 litters/dose group

CLINICAL SIGNS (OFFSPRING)
- small size, bluish colour, blue spot on the neck and eye, scabbing of the right cheek, pale appearance and insufficient milk in the stomach. No relationship with treatment was established for these observations and they were considered to be of no toxicoiogical significance.

BODY WEIGHT (OFFSPRING)
Pup (mean) body weights were in the same range for the control and treated groups.

GROSS PATHOLOGY (OFFSPRING)
- Findings consisted of autolysis of pups found dead at the first Iitter check, scabbing of the right cheek, and insufficient milk in the stomach. No relationship with treatment was established for these observations and they were considered to be of no toxicoiogical significance.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, treatment with Isostearic acid, esters with methyl α-D-glucoside by oral gavage in male and female Wistar Han rats at dose levels of 0, 50, 150 and 1000 mg/kg bw/day revealed parental toxicity at 1000 mg/kg bw/day. No reproduction, breeding and developmental toxicity was observed for treatment up to 1000 mg/kg bw/day.

Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according OECD 422 Isostearic acid, esters with methyl α-D-glucoside in 1% aqueous carboxymethyl cellulose was administered to 10 male and 10 female Wistar Han rats/dose group by daily oral gavage at dose levels of 0, 50, 150, and 1000 mg/kg bw/day. The males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 30 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42 to 44 days). 10 litters per dose group were delivered.

At 1000 mg/kg bw/day statistically significantly reduced haemoglobin, cholesterol and total protein levels (males), and elevated white blood cell counts (determined for only two females) plus alkaline phosphatase levels (males) were found. Increased liver weights (absolute and relative) were noted for high dose males and females.

No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. reproduction, breeding, pup development, and of the adults: mortality, clinical appearance, functional observations, body weight, food consumption, macroscopic and microscopic examination).

The parental NOEL is 150 mg/kg bw/day, based on the findings noted at 1000 mg/kg bw/day

The parental NOAEL is >= 1000 mg/kg bw/day, based on the findings noted at 1000 mg/kg bw/day which were not considered adverse and were without any corroborative findings like histopathological changes.

The reproduction, breeding and developmental NOAEL is >= 1000 mg/kg bw/d.

This Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the rat is acceptable and satisfies the guideline requirements of OECD TG 422.