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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-06-28 to 2020-01-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guidance No. 43 on mammalian reproductive toxicity testing and assessment
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[(2-hydroperoxybutan-2-yl)peroxy]butane-2-peroxol; butane-2,2-diperoxol
EC Number:
700-954-4
Cas Number:
1338-23-4
Molecular formula:
Mixture of C4H10O4 and C8H18O6
IUPAC Name:
2-[(2-hydroperoxybutan-2-yl)peroxy]butane-2-peroxol; butane-2,2-diperoxol

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: S & K-LAP Kft. Császár út 135 2173 Kartal HUNGARY
- Age at study initiation: young, healthy and breeding mature rabbits Females were nulliparous before first insemination at study initiation
- Fasting period before study: no
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 15 - 21°C
- Humidity: 29 - 62 %
- Air changes: 8 - 12 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (sunflower oil). Formulations were prepared in the formulation laboratory of the Test Facility with a frequency and stored according to the results of stability measurements in the frame of the analytical method validation from daily to every three days and stored at room temperature or in the refrigerator.

VEHICLE
- Justification for use and choice of vehicle: the test item is not stable in water or aqueous vehicles. Therefore, sunflower oil was used.
- Concentration in vehicle: 100 mg/mL, 50 mg/mL, 25 mg/mL and 5 mg/mL
- Amount of vehicle: a constant volume of 2 mL/kg bw was administered
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The suitability of the chosen vehicle for the test item was analytically proven. Recovery was 95 % of nominal concentrations at 2 mg/mL and 200 mg/mL in sunflower oil respectively. The test item was proved to be stable in the formulations at least for 24 hours at room temperature and at least 3 days in the refrigerator (5 ± 3°C). Analytical control of dosing solutions (control of test item concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility two times during the study.
Five samples from different places were taken from each concentration for analysis of concentration and homogeneity on two occasions. Similarly, five samples were taken from the vehicle (Control, Group 1) and analyzed.
Details on mating procedure:
- Impregnation procedure: artificial insemination
Day of insemination was regarded as day 0 of gestation. Synchronization of the cycle was completed 48 hours prior to insemination by administering PMSG (gonadotropin) hormone subcutaneously into the neck region. The insemination procedure was performed at the test facility by the breeder.
Duration of treatment / exposure:
From gestation day (GD) 6 to 27
Frequency of treatment:
The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis every day at similar time. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology.
Duration of test:
21 days
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
corresponding to a concentration of 100 mg/mL
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
corresponding to a concentration of 50 mg/mL
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
corresponding to a concentration of 25 mg/mL
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Remarks:
corresponding to a concentration of 5 mg/mL
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
No. of animals per sex per dose:
24 inseminated females per dose group
Control animals:
yes, concurrent vehicle
yes, historical
Details on study design:
- Dose selection rationale:
Justification of the dose selection:
The dose setting was based on findings obtained in the non-GLP preliminary study. According to this dose range finding study the dose level of 300 mg/kg bw/day caused abortion in two of three females after treating with 3 mL/kg treatment volume of sunflower oil up to gestation day 11 and 2 mL/kg from gestation day 12.
At 100 and 300 mg/kg bw/day dose lower (negative) corrected body weight gain was indicated. In this main study two high doses (100 mg/kg bw/day and 200 mg/kg bw/day) were chosen accordingly with the aim to induce some maternal or/and developmental toxicity but not death or severe suffering. The low dose was selected to induce no toxicity. The intermediate dose level of 50 mg/kg bw/day was interpolated geometrically.

- Rationale for animal assignment: random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day, after treatment

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight was recorded on gestation days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28 (accuracy 1 g).
The corrected body weight was calculated for the 28th day of pregnancy (body weight on day 28 minus the weight of the gravid uterus).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes; The food consumption was measured between gestation days 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21, 21 to 24, 24 to 27 and 27 to 28 by re-weighing the non-consumed diet (accuracy 1 g).

WATER CONSUMPTION AND COMPOUND INTAKE: Yes, visual inspection

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 28
- Organs examined: The organs of neck, thorax and abdomen of the does were examined macroscopically. Organs pathological changes which could not be diagnosed macroscopically were fixed in 4 % neutral formaldehyde solution. Corresponding organs from control animals were kept for comparison. Histological examination on organs was not performed. The ovaries and uterus were removed and the uterus (including cervix) of the pregnant females was weighed (accuracy 1 gram). Uterus of each female was examined for early, late embryonic and fetal death and for the number of live fetuses.
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
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
Data were individually recorded on data sheets, transferred, and compiled by computer or compiled manually.
The statistical evaluation of data was performed with the program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant Duncan’s Multiple Range test was used to assess the significance of intergroup differences. If significance is the result of the Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.

Does or litters were excluded from the data evaluation in cases of:
- A disease or death of the doe unrelated to the treatment (total exclusion)

- Non pregnant females i.e. females with no implantation and no corpora lutea (total exclusion)

- Body weight, body weight gain, food consumption, clinical signs and necropsy findings of females with no implantation but corpora lutea i.e. total preimplantation loss (only the intrauterine parameters were evaluated, partial exclusion) - Circumstances unrelated to the test item which are considered to be reason for exclusion, at the discretion of the Study Director Although these animals were excluded from the data evaluation the study report contains all data. A male/female fetus was considered as retarded in body weight/crown-rump length, when its weight/length was below the average minus twofold standard deviation of the control male/female fetuses.
Indices:
Pre-Implantation loss
Number of corpora lutea - Number of implantations / Number of corpora lutea x 100 (%, group mean)

Post-implantation loss
Number of implantation loss – Number of live fetuses / Number of implantations x 100 (%, group mean)


Fetuses

Sex distribution
Number of Male (Female) fetuses / Number of fetuses x 100 (%, group mean)

External abnormalities/litter
Number of fetuses with abnormality / Number of fetuses x 100 (%, group mean)

Visceral abnormalities/litter
Number of fetuses with abnormality / Number of fetuses examined x 100 (%, group mean)

Skeletal abnormalities/litter
Number of fetuses with abnormality / Number of fetuses examined x 100 (%, group mean)
Historical control data:
yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Few amount of faeces was observed on different days in several animals without a dose response. Diarrhea was recorded for one female on G.D. 28 in the 10 mg/kg bw/day group. Noisy breath was observed in three animals in the 50 mg/kg bw/day group 2 to 4 days long and stopped on G.D. 25 the latest as well as in three animals in the 200 mg/kg bw/day group 3 to 9 days long (in one case stopped on G.D. 23 and in two cases on G.D. 28). Considering the lack of clear dose response this was not attributed to the test item, rather to a technical reason by the treatment with oral gavage. Swelling and wound on the muzzle was recorded for one animal in the 50 mg/kg bw/day dose group. This finding disappeared from G.D. 21 and was not judged to be in relationship with the treatment.
These clinical observations were not attributed to be an effect of the test item.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
None of the animals died before scheduled necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant reductions observed in the body weight parameters including corrected body weight/gain. However, between G.D. 6 to 9 and 21 to 24, 24 to 27 as well as 0 to 28 the body weight gain was lower at 200 mg/kg bw/day than in the other groups which cannot be excluded to be a slight effect of the test item. Between G.D. 27 and 28 the body weight gain was statistically significantly (p<0.01) higher in the 100 and 200 mg/kg bw/day and 10 mg/kg bw/day (p<0.05) groups than in the control.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
G.D. 6 to 9 in the 10, 100 and 200 mg/kg bw/day groups (p<0.05) and not at 50 mg/kg bw/day. considering the lack of clear dose response this finding was not associated with the test item. Between G.D. 9 and 12 as well as 24 and 27 the food consumption was lower (-16 and -19%) in the 200 mg/kg bw/day group than in the other groups which cannot completely be ruled out to be due to an effect of the test item.
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:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Observations like pinhead-sized or point-like or pin-prick -sized haemorrhages in the lungs and reddish mottled lungs were seen in the groups. Bloody region of vaginal orifice as well as stomach filled by feed and fetal remains was observed in relationship abortion or early delivery. Anal region was tainted by faeces of one doe with diarrhea in the 10 mg/kg bw/day group. These macroscopic findings were not attributed to the treatment with the test item considering the lack of dose response. Doubled gall bladder was seen in one dam in the control group as a finding unrelated from the treatment. Ca. 10 to 30 mm diameter dark red or/and corrosed-like area was observed in the stomach of three high dose does. In addition one more doe (excluded due to total pre-implantation loss) in this group had similar finding. Considering that this change was seen only in the 200 mg/kg bw/day group, it was attributed to the treatment with the test item and its locally irritating properties.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One female aborted on G.D. 26 in the 50 mg/kg bw/day group and was euthanized on G.D. 27. Considering the lack of dose response, abortion was not attributed to an effect of the test item.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Blood on the tray under the cage was found for one doe in the control group on G.D. 28 and blood/tissue was found for another doe (total post-implantation loss was revealed at Caesarean section) in the 10 mg/kg bw/day group.
There were no statistically significant differences observed in the mean number of corpora lutea, implantation, pre-implantation- and post-implantation loss (including early-, late- and fetal death), total intrauterine mortality (pre- and post-implantation loss summarized).
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no statistically significant differences observed in the mean number of viable fetuses.
Changes in pregnancy duration:
effects observed, non-treatment-related
Description (incidence and severity):
The occurrence of early delivery (GD 27 or 28 (the day before or in the morning of scheduled necropsy) was one in each group including control and excluded 100 mg/kg bw/day. Considering the lack of dose response, early delivery was not attributed to an effect of the test item.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
please refer to details on maternal toxic effects
Other effects:
no effects observed
Details on maternal toxic effects:
The number of inseminated females was 24 in all groups. There were 4 females without any implantation and corpora lutea in the control group and one each in the test item treated groups. The females with corpora lutea but without implantation (total pre-implantation loss) was 1, 0, 2, 1 and 1 in the control, 10, 50 100 and 200 mg/kg bw/day dose groups, respectively. The pregnancy rate was between 79 and 96%, the lowest in the control group. One female in the 10 and two in the 50 mg/kg bw/day dose group had total post-implantation loss. One of these 50 mg/kg bw/day females was excluded from the data evaluation based on a developmental disorder and the other female in this group was excluded due to a disease. One female aborted on G.D. 26 in the 50 mg/kg bw/day dose group and was euthanized. In all groups (except 100 mg/kg bw/day) one female delivered early (on G.D. 27 or to 28 morning).
The number of litters revealed at Caesarean section was 18, 21, 17, 22 and 21 in the control, 10, 50, 100 and 200 mg/kg bw/day dose groups, respectively.
Does or litters listed below were excluded from the data evaluation:
Non pregnant females i.e. females with no implantation:
No implantation and no corpora lutea (total exclusion):
- control: No.: 160, 051, 093, 109
- 10 mg/kg bw/day: 017
- 50 mg/kg bw/day: 012
- 100 mg/kg bw/day: 019
- 200 mg/kg bw/day: 188
No implantation but corpora lutea i.e. total pre-implantation loss (partial exclusion, the intrauterine parameters were evaluated):
- control: No.: 116
- 50 mg/kg bw/day: No.: 130
- 100 mg/kg bw/day: No.: 143
- 200 mg/kg bw/day: No.: 007
Females with total post-implantation loss (partial exclusion, body weight and food consumption parameters excluded)
- 10 mg/kg bw/day: No.: 124
- 50 mg/kg bw/day: No.: 182, 145 (both total excluded, see * and **)
Females with abortion: (excluded: body weight and food consumption data from the day of observed abortion)
- 50 mg/kg bw/day: No.: 115
Females and litters with early delivery: (excluded: body weight and food consumption data of the does from the day of observed early delivery)
- control: No.: 170
- 10 mg/kg bw/day: No.: 095
- 50 mg/kg bw/day: No.: 085
- 200 mg/kg bw/day: No.: 094
A disease of the doe unrelated to the treatment (survived) (total exclusion):
Disease (survived, post-implantation loss):
- 50 mg/kg bw/day: No.: 145 ** (thoracic cavity filled by whitish-yellowish dense phlegma, lungs sticked to pleura)
Developmental disorder of the doe (survived, total post-implantation loss) (total exclusion):
- 50 mg/kg bw/day: No.: 182 * (right kidney absent, left kidney 2-3 fold larger than normal, hypoplastic right uterine horn)

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
necropsy findings

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no significant differences indicated in the fetal growth (body weight and crown-rump length) as well as placental weight (absolute and relative).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
Malformations
The number of affected litters was one in each group including control and except the 10 mg/kg bw/day group.
Spina bifida occulta was observed in one control fetus (No.: 0950055/4). Partial acrania was seen in one fetus (No.:1175196/2) in the 50 mg/kg bw/day group. Exencephaly was found in one fetus (No.: 1456135/3) in the 100 mg/kg bw/day group.
Hyperflexion of one forelimb was seen in one fetus (No.: 1570045/6) in the 200 mg/kg bw/day group. These findings were not attributed to the test item, considering the sporadic distribution in the groups and that failure of neural tube closure occurred also in the control group. Hyperflexion of the forelimbs may occur in control fetuses according to the Historical control data.

Variations
There was no increase in incidence of fetuses and litters with variations (retardation in body weight or crown-rump length). Moreover, the incidence of affected litters with crown-rump length retardation was statistically significantly lower (p<0.005) in the high dose.

Placentas
There were no placental changes found.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There was a statistically significant increase in incidence of fetuses with skeletal abnormalities due to incidence of variations (p<0.05) in the 200 mg/kg bw/day dose group. There were no significant increases indicated investigating the different types of variations observed. There was no increase indicated in the incidence of malformations.

Malformations:
The following skeletal malformations were observed in the fetuses found with malformation at external examination.
The lumbar II arches of fetus No.: 0950055/4 were partially, from lumbar III to sacral I completely unclosed (spina bifida at external examination) in the control group. The skull bones were absent cranial from atlas (brain and facial), the mandible was considered as misshapen and hypoplastic and interrupted in fetus No.:1175196/2 in the 50 mg/kg bw/day group (partial acrania and hypoplastic mandible at external examination).
Interconnecting anterior and posterior fontanelle (dilated suture), hole in parietal; misshapen frontal and parietal; absent interparietal and supraoccipital were found in one fetus (No.: 1456135/3 with exencephaly) in the 100 mg/kg bw/day group. Slight shortness of one side ulna was seen in fetus No.: 1570045/6 (hyperflexion of the forelimb at external examination) in the 200 mg/kg bw/day group.
Sternebral fusions of different degree (fused in a small point, fused by peak, fused) or/and combined with wideness were recorded. Spliss or hole in or between sternebra 5th or 6th or/and xiphoid cartilage were found with low incidences and without a dose response. Multiply malformed ribs or/and vertebra, misshapen cervical vertebra, dumb-bell shaped cartilage and hemicentric ossification of thoracic centrum, multiply malformed vertebrae were found in single cases or/and without a dose response.
When the whole incidence or the single type of skeletal malformations was investigated, there were no significant dose related increases observed, hence the skeletal malformations were not attributed to the treatment.

Variations:
Variations of the skull bones like larger or slightly larger anterior or/and posterior fontanelle or/and slightly dilated suture, slightly incomplete ossification of parietal bone, sternal variations such as less than 5 ossified sternebra, dumb-bell shaped, bipartite or/and asymmetric, misaligned ossification, extra ossification point, fusing tendency, small hole in xiphoid cartilage and slightly bent cartilage, variation of the vertebra, such as bipartite, dumb-bell shaped or asymmetric ossification, ossification delay of pelvic girdle (unossified pubic), talus, pollex, phalanges as well as asymmetric degree of ossification of phalanges of the two sides were recorded.

There was no statistically significant increase indicated in any of these parameters.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the visceral abnormalities/variations/malformations.

Malformations:
There were no malformations found at visceral examinations.

Variations:
Slightly or moderately dilated IIIrd brain ventricle was found in two fetuses in the 100 and two in the 200 mg/kg bw/day groups. This alteration may also occur in control animals according to the Historical data. In one fetus in the 100 mg/kg bw/day group the inter-medial lung lobe was absent. Convoluted- or hydroureter was found in the fetuses with similar incidences in the groups. Acute angled ureter connection to kidney, markedly convoluted and slightly dilated ureter and dilated renal pelvis and doubled gall bladder were found in single cases and not in the high dose group.
The variations found were not attributed to the test item considering the low incidences and/or lack of dose response.
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Basis for effect level:
other: no adverse effects observed up to the highest dose tested

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of this OECD 414 compliant study in rabbits, the NOAEL was determined as follows:
NOAEL (maternal toxicity): 100 mg/kg bw/day
NOAEL (developmental toxicity): 200 mg/kg bw/day
Executive summary:

The test item was examined for its possible prenatal developmental toxicity. Groups of 24 inseminated New Zealand White rabbits were treated with the test item by oral (gavage) administration daily at four dose levels of 10, 50, 100 and 200 mg/kg bw/day respectively from day 6 up to and including day 27 post insemination. A control group of 24 inseminated females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw.


The test item was proved to be stable in sunlflower oil formulations at 2 mg/mL and 200 mg/mL concentration levels for 24 hours at room temperature and at least for three days in the refrigerator (5 ± 3°C) according to the analytical method validation (Study no.: (Toxi-Coop study no. 552-100-4500)) at Toxi-Coop Zrt. The suitability of the chosen vehicle for the test item was analytically proven. Analytical control of dosing solutions was performed during the first and last week of treatment. The mean of the test item concentrations of the test item in the dosing formulations varied in the acceptable range between 90 and 96 % of nominal concentrations at both analytical occasions confirming proper dosing.


During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the does were recorded. The day of insemination was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 28. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed, measured and examined for gross external abnormalities. The placentas were weighed and examined externally.


Fresh visceral examination of each fetus was performed including the determination of the gender.


Head of about 50 % of each litter was removed, fixed in modified Sanomiya solution and washed in 90 % isopropanol. Examination of the heads was done by Wilson's free-hand razor blade method. All skeletons were examined by means of a dissecting microscope after cartilage-bone staining.


There was no test item related mortality, moribund state, abortion or early delivery observed.


The number of litters revealed at Caesarean section was 18, 21, 17, 22 and 21 in the control, 10, 50, 100 and 200 mg/kg bw/day dose groups, respectively.


There were no test item related clinical signs observed. Dark red and/or corrosion- like areas in the stomach revealed at necropsy in the 200 mg/kg bw/day group were attributed to the treatment and irritant properties of the test item.  
Between G.D. 6 to 9 and 21 to 24 24 to 27 as well as 0 to 28 the body weight gain was lower at 200 mg/kg bw/day than in the other groups. Between G.D. 9 and 12 as well as 24 and 27 the food consumption was lower (-16 and -19%) in the 200 mg/kg bw/day group than in the other groups. It cannot completely ruled out that both findings are caused by the test item treatment. There were no treatment related effects observed.
The fetal growth and placental weight were not affected by the treatment. The test item was judged not to increase the incidence of fetal malformations and variations. The summarized incidence of skeletal variations increased statistically significantly (p<0.05) in the high dose group but not the different type of variations. In the lack of a dose- response relationship this observation is not attributed to the test item.