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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
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: Read-across from a guideline study (RL 1) according to OECD TG 422, screening test for reproduction/development toxicity
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:
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: -
- 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 22.2° C)
- Humidity (%): 30 - 70% (actual range: 37 - 94%)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
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.

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
Details on mating procedure:
- M/F ratio per cage: 1/1; one female was cohabitated with one male of the same treatment group
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- 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.
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:
Once daily for 7 days per week
Details on study schedule:
- Parturition: The females were allowed to Iitter normally. Females that were littering were left undisturbed. Offspring was kept with the dam until termination
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
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:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed
on Days 0, 4, 7, 11,14,17 and 20 post-coitum and during lactation on Days 1 and 4.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data NA
Weekly, for males and females. Food consumption was not recorded during the breeding period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and during lactation on Days 1 and 4 post-partum.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect
was suspected.

OTHER:
Reproduction processes:
Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.


Functional Observations:
The following tests were performed in the first five mated males and the first five females with live offspring, from each group:
hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 =normal/present, score 1 = abnormal/absent).
motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring
system; Pearson Technical Services, Debenham, Stowmarket, England).
During the motor activity test, males were caged individually and females were caged with their offspring.
The assigned males were tested during Week 4 of treatment and the assigned females were tested during lactation (all before blood sampling).
In order to avoid hypothermia of pups, dams were removed from the pups for not more than 30-40 minutes.
Sperm parameters (parental animals):
Of the selected 5 males/group of the control and high dose group, additional slides of the testes
were prepared to examine staging of spermatogenesis.
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):
SACRIFICE
- Male animals: All surviving animals -following completion of the mating period (a minimum of 28 days of dose administration)
- Maternal animals: All surviving animals - on lactation Day 5 or shortly thereafter


GROSS NECROPSY
- After sacrifice all parental animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with
special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
- 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.
Statistics:
The following statistical methods were used to analyse the data:
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.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may be rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
No statistical analysis was performed on histopathology findings.
Reproductive indices:
Percentage mating: Number of females mated/ Number of females paired x 100
Fertility index: Number of pregnant females/ Number of females paired x 100
Conception rate: Number of pregnant females/ Number of females mated x 100
Gestation index: Number of females bearing live pups/ Number of pregnant females x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
Percentage live females at First Litter Check: Number of live female pups at First Litter Check/ Number of live pups at First Litter Check x 100
Percentage of postnatal loss days 0-4 post partum: Number of dead pups on Dav 4 post partum/ Number of live pups at First Litter Check x 100
Viability index: Number of live pups on Day 4 post partum/ Number of pups born alive x 100

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:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
No toxicologically-relevant clinical signs were noted up to 1000 mg/kg.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period. Food consumption before
or after allowance for body weight was similar between treated and control animals.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): NA

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS) no data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The spermatogenic staging profiles were normal for all Group 1 and Group 4 males evaluated.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Reproduction parameters were unaffected by treatment up to 1000 mg/kg body weight/day.
All pairs mated within four days and all females were pregnant.
No treatment related findings were observed for mating performance, fertility parameters, gestation duration, number of dead and living pups at first
litter check, number of implantation sites and number of corpora lutea.
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.

ORGAN WEIGHTS (PARENTAL ANIMALS)
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 PATHOLOGY (PARENTAL ANIMALS)
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
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.

OTHER FINDINGS (PARENTAL ANIMALS):
Functional observations: Hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity were normal in the selected animals.

For further details on haematology and clinical biochemistry please refer to chapter: 7.5.1 "Repeated dose toxicity: oral".

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: The described findings at 1000 mg/kg bw were not considered adverse and were without corroborative findings.

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)
Postnatal loss and viability index were similar for the control and treated groups.

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.
Development of pups was unaffected by treatment up to 1000 mg/kg body weight/day.


Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
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).

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
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/Develpmental Toxicity Screening Test according OECD 422 test substance Isostearic acid, esters with methylα-D-glucoside (100% UVCB-Substance (80% Methyl Glucoside Isostearate Esters (mainly Di-), 16% Isostearic Acid, 4% Methyl Glucoside (not soluble in Olive Oil)) 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).

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. These changes were noted without corroborative findings (e.g. histopathological findings). However, all values are in the range of the laboratory´s historical control data and therefore considered to be of no toxicological relevance.

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, breeding and pup development).

In conclusion the findings noted at 1000 mg/kg were not considered adverse and were without corroborative findings, the parental No Observed Adverse Effect Level (NOAEL) was established to be 1000 mg/kg body weight/day.

No treatment-related changes were noted for reproduction, breeding and pup development, therefore a NOAEL of ≥ 1000 mg/kg bw/d was established for reproductive and developmental toxicity.

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422) in rat.