A mixture of: 2-ethylhexyl mono-D-glucopyranoside; 2-ethylhexyl di-D-glucopyranoside

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February-October 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed and reported study according to standard/guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

.alpha.-D-Glucopyranoside, 2-ethylhexyl

Cas Number:

125590-73-0

IUPAC Name:

.alpha.-D-Glucopyranoside, 2-ethylhexyl

Constituent 2

Reference substance name:

A mixture of: 2-ethylhexyl mono-D-glucopyranoside; 2-ethylhexyl di-D-glucopyranoside

EC Number:

414-420-0

EC Name:

A mixture of: 2-ethylhexyl mono-D-glucopyranoside; 2-ethylhexyl di-D-glucopyranoside

Cas Number:

125590-73-0

Molecular formula:

(C6H10O5)n, C8H18O n = 1,2

IUPAC Name:

Reaction mass of 2-ethylhexyl mono-D-glucopyranoside and 2-ethylhexyl di-D-glucopyranoside

Details on test material:

Name of test compound: SPE 93279
Batch no.: 3001036216
Appearance: Liquid
Purity: solids: ca. 64.7%, water 35.3%
Storage: at room temperature (20±5 degr. C), away from direct sunlight
Expiry date: 31 October 2005
Stability in the vehicle: stable for at least 4 h

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Fuellinsdorf Switzerland
- Age at study initiation: 11 weeks (at pairing)
- Weight at study initiation: 191-234 g
- Fasting period before study: not applicable
- Housing: individually in type-3 Macrolon cages
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): target 22±3
- Humidity (%): target: 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From:26 February (delivery of the animals) To: 24 March 2005

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a glass beaker on a tared precision balance and the vehicle (Milli-Q-Water) added (wlv). The vehicle was heated to approx. 30°C before addition to the test item to dissolve crystal formation in the formulation. Using an appropriate
homogenizer a homogenous mixture was prepared. Having obtained a crystal-free, homogenous mixture (determined visually), the dose formulations were left at room temperature until dosing. During the daily administration period homogeneity of the test item in
the vehicle was maintained using a magnetic stirrer.
Dose formulations were prepared daily, no longer than 4 hours prior to dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): water was used
- Concentration in vehicle: not indicated
- Amount of vehicle (if gavage): 5 mL/kg
A correction factor of 1.55 was used for the preparation of the dose formulations to adjust for the purity of the test compound.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for determination of concentration, homogeneity and stability (4 hours) of the dose formulations were taken during the first week of the administration period. Additionally, samples for determination of concentration and homogeneity were taken during the
last week of the administration period. On each occasion three samples of approximately 2 g were taken from the top, middle and
bottom of each formulation and transferred into flat bottomed flasks. Stability samples were taken from the middle only. The samples were frozen (-25°C to -15°C) pending analysis. Samples were sent on dry ice to Dr. D. Flade, RCC Ltd, Environmental Chemistry &
Pharmanalytics, CH-4452 ltingen / Switzerland. Analysis was performed using LC/MS (although in the main part of the report it was
indicated that analyses had been performed using HPLC).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: animals were housed in special automatic mating cages (with synchronized timing to initiate the nightly
mating period), until evidence of copulation
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. No info.
- Further matings after two unsuccessful attempts: no info
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy:vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

from day 6 to day 20 (inclusive) post coitum

Frequency of treatment:

daily

Duration of test:

21 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

22 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on RF study using 0, 50, 250 and 600 mg/kg bw/day. 600 mg/kg resulted in 60% (3/5) mortality
- Rationale for animal assignment (if not random): mated rats were assigned to the different groups using a computer-generated random algorithm
:

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: daily from day 0 to day 21 post coitum

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: at 3-day intervals

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: all internal organs
Post mortem examination, including gross macroscopic examination of all internal organs, with emphasis on the uterus, uterine
contents, position of fetuses in the uterus and number of corpora lutea, was performed and the data recorded. The uteri (and
contents) of all females with live fetuses were weighed at necropsy on day 21 post coitum to enable the calculation of the
corrected body weight gain. From all females the following organs were preserved in neutral phosphate buffered 4% formaldehyde solution for possible future examination: trachea, lungs, heart and liver.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:

Fetal examinations:

- External examinations: Yes (all per litter)
- Soft tissue examinations: Yes (half per litter)
- Skeletal examinations: Yes (half per litter)
- Head examinations: Yes ( half per litter)

Statistics:

The following statistical methods were used to analyse body weights, food consumption, reproduction and skeletal examination
data:
- Means and standard deviations of various data were calculated.
- If the variables could be assumed to follow a normal distribution, the Dunnett many-one t-test, based on a pooled variance
estimate, was used for intergroup comparisons (i.e. single treatment groups against the control group).
- The Steel test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.
References:
C.W. Dunnett A Multiple Comparison Procedure for Comparing Several Treatments with a Control,
J. Amer. Statist. Assoc. 50, 1096-1 121 (1 955).
R. G. Miller Simultaneous Statistical Inference, Springer Verlag, New York (1981).
R.A. Fisher Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1 950).

Indices:

Pre and post-implantation losses

Historical control data:

Included in the report

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical signs: in the high dose group, one female (No. 85) was found dead on day 18 post coitum. All other females survived until
scheduled necropsy. For two females in this group, rales were noted on single days of the treatment period. Although it was an
isolated mortality and the incidence of this clinical sign was very low, both were considered to be test item related, since mortalities and rales have been repeatedly noted in other rat studies with this test item.
Food intake: in the high dose group, mean food consumption was transiently slightly reduced after the onset of treatment. After the first six days of treatment mean food consumption recovered to values similar to those of the vehicle control.
Body weight: mean absolute body weights and body weight gain were not affected by treatment with the test item at any dosage.
Mean corrected body weight gain (corrected for gravid uterus weight) was similar in all groups and gave no indication of a test item-related effect.
Reproduction data: The relevant reproductive parameters (incidence of post-implantation loss and number of fetuses per dam)
were similar in all groups and gave no indication of a test item-related effect.
Necropsy: During macroscopic examination no findings that were considered to be test item-related were noted.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day

Basis for effect level:

clinical signs

necropsy findings

other: maternal toxicity

Results (fetuses)

Fetal body weight changes:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

During skeletal examination of the fetuses abnormal findings were noted in:

14 out of 124 examined fetuses (in 10 litters) in the control group

11 out of 114 examined fetuses (in 10 litters) in the low dose group

5 out of 136 examined fetuses (in 4 litters) in the mid dose group

15 out of 124 examined fetuses (in 10 litters) in the high dose group.

In the high dose group, a marginal incidence of wavy ribs (3 fetuses affected compared with 0 fetuses in the vehicle control) was noted. Since this incidence of wavy ribs was in the range of historical control data this finding was considered to reflect the range of natural variability. The findings noted mainly comprised common findings such as fused zygomatic arches and displaced pelvic girdles. Neither type nor frequency of these abnormal findings was considered to be test item-related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

During visceral examination of fetuses abnormal findings were noted in:

52 out of 135 examined fetuses (in 21 litters) in the control group

42 out of 122 examined fetuses (in 19 litters) in the low dose group

53 out of 149 examined fetuses (in 18 litters) in the mid dose group

53 out of 137 examined fetuses (in 20 litters) in the high dose group.

Types and incidences of abnormal findings (such as lenticular lesion of the eye, elongated thymus, thinned diaphragm, left sided umbilical artery and displaced testis) in the low, mid and high dose groups were similar to those noted in the vehicle control and gave no indication of test item-related effects. In the high dose group, one fetus (No. 540) with multiple cranial findings and one fetus (No. 91) with situs inversus, displaced kidneys and abnormal lung lobulation were noted. These isolated findings were considered to be not test item-related. In all four groups, discoloured head regions were observed, which were most probably fixation artefacts.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no treatment-related embryotoxic effects (see below for a complete summary).

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Effect level:

>= 500 mg/kg bw/day

Basis for effect level:

changes in sex ratio

external malformations

skeletal malformations

visceral malformations

other: developmental toxicity

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

not specified

Any other information on results incl. tables

SEX RATIOS

No test item-related effects on the sex ratio of fetuses were noted in any group, which were always close to 50% each.

BODY WEIGHTS

Mean fetal body weights were similar in all groups and gave no indication of a test item-related effect.

EXTERNAL EXAMINATION

No abnormalities were noted during external examination of fetuses. In the high dose group, agnathia inferior, astomia and reduction or absence of the eyes were noted for one fetus (No. 540, dam 73). This isolated finding was considered to be incidental.

VISCERAL EXAMINATION (MICRODISSECTION TECHNIQUE)

During visceral examination of fetuses abnormal findings were noted in:

52 out of 135 examined fetuses (in 21 litters) in the control group

42 out of 122 examined fetuses (in 19 litters) in the low dose group

53 out of 149 examined fetuses (in 18 litters) in the mid dose group

53 out of 137 examined fetuses (in 20 litters) in the high dose group.

Types and incidences of abnormal findings (such as lenticular lesion of the eye, elongated thymus, thinned diaphragm, left sided umbilical artery and displaced testis) in the low, mid and high dose groups were similar to those noted in the vehicle control and gave no indication of test item-related effects. In the high dose group, one fetus (No. 540) with multiple cranial findings and one fetus (No. 91) with situs inversus, displaced kidneys and abnormal lung lobulation were noted. These isolated findings were considered to be not test item-related. In all four groups, discoloured head regions were observed, which were most probably fixation artefacts.

ABNORMAL FINDINGS FROM FETAL SKELETAL EXAMINATION

During skeletal examination of the fetuses abnormal findings were noted in:

14 out of 124 examined fetuses (in 10 litters) in the control group

11 out of 114 examined fetuses (in 10 litters) in the low dose group

5 out of 136 examined fetuses (in 4 litters) in the mid dose group

15 out of 124 examined fetuses (in 10 litters) in the high dose group.

In the high dose group, a marginal incidence of wavy ribs (3 fetuses affected compared with 0 fetuses in the vehicle control) was noted. Since this incidence of wavy ribs was in the range of historical control data this finding was considered to reflect the range of natural variability. The findings noted mainly comprised common findings such as fused zygomatic arches and displaced pelvic girdles. Neither type nor frequency of these abnormal findings was considered to be test item-related.

SKELETAL EXAMINATION OF FETUSES - STAGE OF DEVELOPMENT AND COMMON VARIANTS

Examination of the stage of development revealed that the right and left toes (toes 2, 3 and 4, proximal phalanx, respectively) were statistically significantly less ossified in the high dose group than in the vehicle control both calculated on a litter and fetus basis. However, this finding was

considered not to be test item-related given that, in control fetuses, ossification of these structures, as judged by both the extent and depth of alizarin staining, was poor, and there were no other findings that were suggestive of delay in ossification.

CARTILAGE EXAMINATION OF FETUSES (ABNORMAL FINDINGS AND COMMON VARIANTS)

During cartilage examination of the fetuses abnormal findings were noted in:

0 fetuses (in 0 litters) in the control group

2 fetuses (in 2 litters) in the low dose group

0 fetuses (in 0 litters) in the mid group 3

1 fetus (in 1 litter) in the high dose group.

The findings noted were common cartilaginous abnormalities such as offset costal cartilages, cervical vertebra with additional ventral plate and fused lumbar vertebral bodies. Neither the types nor the frequencies of these findings gave any indication of test item-related effects. When calculated on a litter basis, the incidence of branched xiphoid cartilage in the low dose group was statistically significantly lower than in the vehicle control. When calculated on a fetus basis, the incidence of a longer costal cartilage in the low dose group was statistically significantly higher. Both

incidences of common variations were considered to be of no toxicological relevance.

Applicant's summary and conclusion

Conclusions:

Based on the results of the present study, the NOEL (no observed effect level) for maternal toxicity was considered to be 250
mg/kg body weight/day, the NOEL for developmental toxicity was 500 mg/kg body weight/day.

Executive summary:

The purpose of this study was to assess the effects of SPE 93279 on the pregnant female and on embryonic and fetal development when administered orally by gavage once daily to mated female rats from day 6 through to day 20 post coitum, inclusive. Each group consisted of 22 mated female rats. SPE 93279 was administered once daily at dose levels of 0, 50, 250 and 500 mg/kg body weight/day. A standard dose volume of 5 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (Milli-Q-Water). All surviving females were sacrificed on day 21 post coitum and the fetuses were removed by Caesarean section. Examination of dams and fetuses was performed in accordance with international recommendations.

Maternal toxicity:

At 500 mg/kg body weight/day, one female (No. 85) was found dead on day 18 post coitum. All other females survived until scheduled necropsy. For two females at 500 mg/kg body weight/day rales were noted on single days of the treatment period. Although it was an isolated mortality and the incidence of this clinical sign was very low, both were considered to be test item-related, since mortalities and rales have been repeatedly noted in other rat studies with this test item. At 500 mg/kg body weight/day, mean food consumption was transiently slightly reduced only after the onset of treatment. Mean food consumption at 50 and 250 mg/kg body weight/day was not affected by treatment with the test item. No test item-related effects on body weight gain were noted. The relevant reproductive parameters (incidence of post-implantation loss and number of fetuses per dam) were similar in all groups and gave no indication of a test item-related effect. There were no treatment-related changes at necropsy.

Fetal toxicity:

Mean fetal body weights were similar in all groups and gave no indication of a test item-related effect. No abnormalities were noted during external examination of fetuses. No indication of test item-related effects was noted during the visceral examination. During skeletal examination, no findings that were considered to be test item-related were noted.

Overall, treatment with the test item at 500 mg/kg body weight/day resulted in rales in two females and in the death of one animal. Since mortalities and rales were also noted in other rat studies, including the RF study at 600 mg/kg bw, both were considered to be test item-related. Based on these results the NOEL (no observed effect level) for maternal toxicity was considered to be 250 mg/kg body weight/day, the NOEL for developmental toxicity was established at the high dose level of 500 mg/kg body weight/day.