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Toxicological information

Toxicity to reproduction

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Endpoint:
toxicity to reproduction
Remarks:
other: combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-09-25 to 2008-11-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
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:
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

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Wistar Han (Crl:WI(Han))
Sex:
male/female
Details on test animals and environmental conditions:
Accommodation offspring
- Offspring was kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1 % aqueous carboxymethyl cellulose (Genfarma, Zaandam, The Netherlands).
Details on exposure:
After acclimatisation, four groups of ten male and ten female Wistar Han rats were exposed by
oral gavage to the test substance at 0, 50, 150, and 1000 mg/kg/day.
Males were exposed for 30 days, i.e. 2 weeks prior to mating, during mating, and up to
termination. Females were exposed for 42 to 44 days, i.e. during 2 weeks prior to mating, during
mating, post-coitum, and during at least 4 days of lactation.
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:
Quantitative analysis was based on the analytical method validated for the test substance in NOTOX project 488541.
Preparation of formulations was considered acceptable if the mean accuracy was in the range 85% - 115% of the target concentration and if the coefficient of variation was <= 10%. Formulations were considered stable if the relative difference between the stored and freshly taken samples
was <= 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:
Daily treatment
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 / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
oral gavage
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
oral gavage
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
oral gavage
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
no positive control group

Examinations

Parental animals: Observations and examinations:
At 1000 mg/kg, clinical laboratory investigations revealed statistically significantly reduced haemoglobin, cholesterol and total protein levels (males), and statistically significantly elevated white blood cell counts (females; only for two determined) and alkaline phosphatase levels (males). Increased liver weights (absolute and relative) were noted for high dose males and females. These changes were noted without corroborative findings (e.g. histopathological findings).
No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, clinical appearance, functional observations, body weight, food consumption, macroscopic and microscopic examination, reproduction and breeding).
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.
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 01 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):
There were no unscheduled deaths.
Macroscopic Findings

Macroscopic examinations were performed of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. All macroscopic findings were considered to be spontaneous in nature and did not distinguish treated animals from the controls.

Microscopic Findings
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.
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.
Reproductive indices:
Breeding parameters were unaffected by treatment up to 1000 mg/kg body weight/day.
Postnatal loss and viability index were similar for the control and treated groups.
Offspring viability indices:
Breeding parameters were unaffected by treatment up to 1000 mg/kg body weightlday.
Postnatal loss and viability index were similar for the control and treated groups.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, haemoglobin levels were slightly reduced for males (statistical significant at 5%) and white blood cell counts (WBC) were higher for females (statistical significant at 1%; only determined for two females). A statistical significant reduction in prothrombin time (PT) was noted for males at 150 mg/kg, but in the absence of a dose-dependent trend it was considered to be of no toxicological relevance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, males showed elevated alkaline phosphatase (ALP) levels (statistical significant at 1%), and significantly reduced levels of cholesterol and total protein compared to vehicle controls (both statistical significant at 5%).
Aspartate aminotransferase (ASAT) levels showed a reduction for females at all treatment levels without a dose response relationship. However, for this parameter, the values for the concurrent control animals were much higher than data from historical controls (historical control mean = 66.6, Group 1 mean = 121.4), and as such, the reduced ASAT levels were not considered to be toxicologically relevant. The cause of these increased values for the concurrent control group was unclear. However, as no corroborative findings were noted it was not considered toxicological relevant.
Chloride levels were slightly lower for females at 150 and 1000 mg/kg. However, as this change was very slight and within the normal range, it was not considered toxicologically relevant. All other statistically significant changes from controls (decreased glucose levels at 50 mg/kg and increased inorganic phosphate values at 150 mg/kg, both for males) were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity were normal in the selected animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, higher absolute liver weights and liver to body weight ratios were observed for both sexes (not statistically significant for absolute liver weights of the males).
At 150 mg/kg, increased testes weights (absolute and body weight ratio) were noted and an increase in seminal vesicle weight (body weight ratio) was seen at 50 mg/kg. These findings in both treatment groups were considered not to be a sign of toxicity as no dose response relationship was noted.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Incidental findings included foci on the stomach glandular mucosa, pelvic dilation of the kidneys, reduced size of the seminal vesicles, ectopic splenic tissue, a bent tail apex, and alopecia of several body parts. These findings are occasionally seen among rats used in these types of studies. As they remained within the range of biological variation for rats of this age and strain, these findings were considered to be changes of no toxicological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All recorded microscopic findings were within the range of background pathology encountered in Wistar Han 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.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Reproduction parameters were unaffected by treatment up to 1000 mg/kg body weight/day.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Reproduction parameters were unaffected by treatment up to 1000 mg/kg body weight/day.
Reproductive performance:
no effects observed
Description (incidence and severity):
Reproduction parameters were unaffected by treatment up to 1000 mg/kg body weight/day.

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)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
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

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
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.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Pup (mean) body weights were in the same range for the control and treated groups.
Food consumption and compound intake (if feeding study):
not examined
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:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
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.
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
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: : 100% , 50 mg/kg bw: : 100%, 150 mg/kg bw: : 99.2%, 1000 mg/kg bw: : 98.4% (significant at 5 %)
- Dead pups at first litter check: 0 mg/kg bw: 3 males, 2 females; 50 mg/kg bw: 1 male, 1 female, 150 mg/kg bw: 0; 1000 mg/kg bw: 1 female
- Dead pups post natal: at 0, 50 mg/kg bw: 0, at 150 mg/kg bw: 1 male, at 1000 mg/kg bw: 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.


Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No adverse effects were observed

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 50, 150 and 1000 mg/kg body weight/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 test substance 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.