Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994
Reference Type:
publication
Title:
Evaluation of the pre-, peri-, postnatal toxicity of monoethanolamine in rats following repeated oral administration during organogenesis
Author:
Hellwig J and Liberacki AB
Year:
1997
Bibliographic source:
Fundam Appl Toxicol.; 40(1):158-62.

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): monoethanolamine, Substance No. 91 / 351
- Physical state: Liquid/colorless
- Analytical purity: 100 %
- Production date: August 01 , 1991
- Lot/batch No.: Sample 25 (cont . production)
- Storage condition of test material: To be stored under exclusion of oxygen at room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Sexually mature, virgin Wistar rats (Chbb :THOM (SPF)) supplied by Karl THOMAE, Biberach an der Riss, Germany,
- Age at study initiation: 60 days
- Weight at study initiation: mean weight approx. 223.7 g
- Fasting period before study: none
- Housing: singly in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm²) ;
- Diet: ground Kliba 343 feed rat/mouse/hamster supplied by Klingentalermuehle AG, Kaiseraugst, Switzerland ad libitum
- Water: tap water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Each day the test substance solutions were freshly prepared shortly before the test substance was administered. For the preparation of the solutions, an appropriate amount of the test substance was weighed in a volumetric flask and subsequently topped up with doubly distilled water and intensively shaken.

VEHICLE
- Concentration in vehicle:
- Amount of vehicle (if gavage): 10 ml/ kg bw.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity of the test substance was proven by visual inspection. The content of active ingredient was 99 .4% before the beginning of the study. The reanalysis of the test substance proved its stability (content: 99 .5%)
Details on mating procedure:
- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: [yes]
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
Duration of treatment / exposure:
day 6 - 15 of gestation
Frequency of treatment:
daily, once per day
Duration of test:
up to day 21 of gestation
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 40, 120, 450 mg/kg bodyweight
Basis:
actual ingested
No. of animals per sex per dose:
40 dams per dosing group
Control animals:
yes, concurrent vehicle
Details on study design:
On day 0, the animals were assigned to the different test groups according to a randomization plan. The test substance was administered to the animals orally (by gavage) once a day during the period of major organogenesis (day 6 to day 15 p .c .) always at approx. the same time of day (in the morning). The animals of the control group were treated in the same way with the vehicle (doubly distilled water). The volume administered each day was 10 ml/kg body weight. The calculation of the volume administered was based on the individual body weight determined at the beginning of the administration period (day 6 p.c.). On day 20 p.c., the first 25 animals/group were sacrificed in a randomized order and examined macroscopically. The fetuses were dissected from the uterus and further investigated with different methods. The other animals (15/group) were allowed to litter and rear their pups up to day 21 p.p. (post partum). On day 21 post partum (p.p.) or one of the following days the relevant dams and pups were sacrificed and examined macroscopically.

Examinations

Maternal examinations:
Clinical examinations
Food consumption
With the exception of day 0 p.c. (all animals) and days 0 p.p. and 21 p.p. (for animals with terminal
sacrifice on day 21 p.p. only), the consumption of food was determined on the same days as was body weight. Food consumption was not determined for the females without litter during the lactation period of the dams used in parallel.

Body weight data
All animals were weighed on days 0, 1, 3, 6, 8, 10, 13, 15, 17 and 20 p.c. Body weights of the animals with
terminal sacrifice on day 21 p.p. were additionally determined on the day of birth and on days 4, 7, 14 and
21 p.p. The body weight change of the animals was calculated from these results. Body weights of the animals without litter were not determined during the lactation period of the dams used in parallel.

Corrected body weight gain (net maternal body weight change)
Furthermore, the corrected body weight gain was calculated for all animals with terminal sacrifice on day 20 p.c. (terminal body weight on day 20 p.c. minus weight of the uterus before it was opened minus body
weight on day 6 p.c.).

Clinical symptoms
All animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited. The nesting, littering, and lactation behavior of the dams with terminal sacrifice day 21 p.p. was generally evaluated in the mornings in connection with the daily clinical inspection of the dams. Only if there were any special findings (e.g., animal could not litter, umbilical cord not cut), these specific findings were documented with the dam concerned.

The littering behavior of the relevant dams was also inspected on weekdays (except holidays) in the afternoons in addition to the evaluations in the mornings. These reevaluation were documented separately, but, as before, findings were only recorded with the dams concerned. Moreover, the duration of gestation, the number of live and dead pups at birth and litter size were recorded for the animals with terminal sacrifice on day 21 p.p. For these animals the fertility and the gestation indices were calculated according to the following formulae :

fertility index = (n pregnant animals/ n mated animals) x 100

gestation index = (n animals with litters/ n pregnant animals) x 100

The values listed in the Summary Tables are group means determined from the fertility/gestation indices of the individual animals.

Mortality
A check was made twice a day on working days or once a day (Saturday, Sunday or on public holidays).
Ovaries and uterine content:
Examinations of the dams at termination
Dams with terminal sacrifice on day 20 p.c.
On day 20 p.c., the dams were sacrificed in randomized order by cervical dislocation and the fetuses dissected from the uterus. After the dams had been sacrificed , they were necropsied and assessed by gross pathology. The uterus and the ovaries were removed and the following data were recorded :

Weight of uterus before it was opened
- Number of corpora lutea
- Number and distribution of implantation sites classified as :
• live fetuses
• dead implantations:
a) early resorptions (only decidual or placental tissues visible or according to Salewski from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy)
b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been
opened)

Furthermore, calculations of conception rate and pre- and postimplantation losses were carried out:
- The conception rate (in %) was calculated according to the following formula :

conception rate = (number of pregnant animals/ number of fertilized animals) x 100

- The preimplantation loss (in % ) was calculated according to the following formula:

((number of corpora lutea - number of implantations)/number of corpora lutea) x 100

- The post implantation loss (in % ) was calculated from the following formula:

((number of implantations - number of live fetuses)/number of implantations) x 100

Dams with terminal sacrifice on day 21 p.p.
On day 21 post partum (p. p. ) the relevant dams were sacrificed by cervical dislocation. After the dams had been sacrificed, the following examinations were carried out:
- gross - pathological examination
- staining of uterus according to Salewski for determination of the number of implantations

Furthermore, calculations of conception rate and postimplantation loss were carried out:
- The conception rate (in %) was calculated according to the following formula :

conception rate = (number of pregnant animals/ number of fertilized animals) x 100

- The post implantation loss (in % ) was calculated from the following formula:

((number of implantations - number of live fetuses)/number of implantations) x 100
Fetal examinations:
Examination of the fetuses:
Examination of the fetuses after dissection from the uterus
At necropsy each fetus was weighed, sexed and examined macroscopically for any external findings. The sex was determined by observing the distance between the anus and the base of the genital tubercle and was later confirmed in all fetuses fixed in Bouin's solution by internal examination. If there were discrepancies between the "external" and the "internal" sex of a fetus, the fetus was finally sexed according to the appearance of its gonads. Furthermore, the viability of the fetuses and the condition
of the placentae, the umbilical cords, the fetal membranes and fluids were examined. Individual placental
weights were recorded. After these examinations, approximately one half of the fetuses per dam was placed in ethyl alcohol and the other half was placed in Bouin's solution for fixation and further evaluation.

Soft tissue examination of the fetuses
After fixation in Bouin's solution, approximately one half of the fetuses of the dams of all groups was examined for any findings in the organs according to the method of Barrow and Taylor with special attention being paid to the kidneys and the ureters. After the examination, these fetuses were discarded with the exception of the kidneys, which were placed into cassettes separately for each fetus and kept in 4% formaldehyde solution for possible further examination by light microscopy. Moreover, after fixation of the fetuses placed in ethyl alcohol for further evaluation of the fetal skeletons the organs of these fetuses
were examined macroscopically. Thereafter, the kidneys of each fetus were placed into cassettes and kept in 4% formaldehyde solution for a possible further examination by light microscopy, while the other organs were discarded. Afterwards the carcasses of these fetuses were stained according to a modified method (Dawson) for the presentation of the skeletons.

Skeletal examination of the fetuses
After fixation in ethyl alcohol and examination of the organs, the skeletons of the fetuses were stained according to a modified method of Dawson. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After these examinations the relevant fetuses were retained by litter.

Evaluation criteria for assessing skeletons and organs of the fetuses
In the present investigations the following terms (definitions) were used for describing a change:
- Malformations (concerning external, soft tissue and skeletal observations)
Rare and/or probably lethal changes were classified as malformations (e.g. exencephaly, atresia ani, hernia umbilicalis).

- Variations (concerning external, soft tissue and skeletal observations)
Changes which occur regularly also in control groups and have generally no adverse effect on survival were regarded as variations (e.g. dilated renal pelvis).

- Retardations (concerning skeletal observations only)
Delays in skeletal development compared with the norm at the time of the examination were considered to be retardations (e.g. sternebra(e) not ossified)

- Unclassified observations (concerning external and soft tissue observations, only)
External or soft tissue observations, which could not be classified as malformations or variations (e.g.
blood coagulum around placenta).

Examination of the pubs:
Pup number and status at delivery
All pups derived from the females were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn members of each litter. Pups which died before the first determination of their status on the day of birth were designated as stillborn pups.

Pup viability / mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods which will be described in detail before.
The number and percentage of dead pups on the day of birth (day 0) and of pups dying between days 1-4, 5-7, 8-14 and 15-21 of the lactation period were determined; however, pups which died accidentally were
not included in these calculations. The number of live pups/litter was calculated on the day of birth, and on
lactation days 4, 7, 14 and 21. Furthermore, viability and lactation indices were calculated according to the
following formulas :

Viability index (%) = (number of live pups on day 4 after birth/ number o f liveborn pups on the day of birth)
x 100

Lactation index (%) = (number of live pups on day 21 after birth/ number of live pup s on day 4 after birth)
x 100

Sex ratio
On the day of birth (day 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. During the following time the sex of the pups was assessed by the external appearance of the anogenital region and/or the mammary line of the animals and was finally confirmed at necropsy. The sex ratio was calculated for day 0 and day 21 after birth according to the following formula:

sex ratio = (number of live male or female pups on day 0/21 / number of live male and female pups on day 0/21) x 100

Pup body weight data
The pups were weighed on the day after birth (day 1 p.p.) and on days 4, 7, 14 and 21 after birth.
Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). In the relevant summary tables pup body weights and pup body weight gains are listed for males, females and males + females.

Pup clinical observations
The pups were examined each day for clinical symptoms (including gross-morphological findings).

Pup necropsy observations
After sacrifice on day 21 p.p. or one of the following days (by means of CO2) or intercurrent death, the pups were examined externally, eviscerated and their organs were assessed macroscopically with special attention being paid to the urinary tract. After the macroscopic examination of the pups, the kidneys of each pup were placed into cassettes and fixed in 4% formaldehyde solution for a possible further examination by light microscopy. If there were notable findings or if abnormalities were found in the daily clinical observation of the animals after their delivery, the affected animals were, if it was deemed necessary, examined additionally using appropriate methods (e.g., skeletal staining according to modified Dawson's method and/or further processing of head according to Wilson's method. The stained skeletons were evaluated under a stereomicroscope or a magnifying glass. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation (with the exception of the kidneys (see above)).
Statistics:
Dunnett-Test was used for a simultaneous comparison of several dose groups with the control. The hypothesis of equal means was tested. This test was performed two-sided and was used for the statistical evaluation of food consumption, body weights and body weight change (females and pups), corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, number of corpora lutea, number of implantations, number of resorptions and number of live fetuses; proportion of preimplantation loss, postimplantation loss, resorptions and live fetuses in each litter; litter mean fetal body weight and litter mean placental weight, duration of gestation and number of pups delivered per litter. For the body weight and the body weight change of the pups the mean weight of each litter was used for the statistical analysis (statistical unit = litter).
Fisher' s Exact Test was used for a pairwise comparison of each dose group with the control for the hypothesis of equal proportions. This test was performed one - sided was used for statistical evaluation of the following parameters: female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings, female fertility index, gestation index, females with liveborn, stillborn and with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy.
The Wilcoxon Test was used for a comparison of each dose group with the control for the hypothesis of equal medians. This test was performed one-sided and was used for the proportion of fetuses with malformations, variations, retardations and/or unclassified observations in each litter and for the proportion of affected pups per litter with necropsy observations. If the results of these tests were significant, labels (*for ≤ 5 0.05, ** for p ≤ 0.01) were printed in the summary tables.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Monoethanolamine caused some signs of maternal toxicity when administered by gavage to pregnant Wistar rats from days 6 - 15 of gestation at the highest dose level tested, 450 mg/kg/day. Maternal toxicity was substantiated by reduced food consumption, lower mean body weights and impaired body weight gain. The oral administration of monoethanolamine at 450 mg/kg/day or doses below this had no influence on resorption rate, number of live fetuses or pups/dam, mean fetal weight or pup body weights.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No signs of developmental toxicity occurred up to and including the highest dose level (450 mg/kg/day), especially no substance-induced teratogenic effects were observed neither in the fetuses nor in the pups. Furthermore, there were no indications for any substance-related growth retardations. The urinary tract of the rat fetuses/pups did not show any treatment-related findings. Dilated renal pelvis and/or hydroureter were found in a considerable, but according to historical control data, not unexpected high number of fetuses of all groups including the controls without any relation to dosing, but did not occur at an increased rate in the pups of the substance-related groups. All skeletal malformations, variations, or retardations which occurred did not show a clear dose-response relationship, can be found at comparable or even higher rates within the historical control and/or the differences between the groups are without biological significance.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 450 mg/kg bw/day
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Results summary:

The following findings were obtained and assessed as substance-related:

Test group 3 (450 mg/kg body weight/day):

- statistically significantly reduced food consumption at the beginning of the treatment period (days 6 -

8 p.c.), the final days of the gestation period (days 17 - 20 p.c.) and during the first days of the

lactation period (days 0- 4 p.p.).

- statistically significantly lower mean dam body weights than the controls on days 15, 17 and 20 p.c. and on days 0, 4, 7 and 21 p.p. ; impaired body weight gain of the dams during posttreatment days 15 - 20 p.c.

 

Test group 2 (120 mg/kg body weight/day) :

- no substance-related effects on dams, fetuses or pups

 

Test group 1 (40 mg/kg body weight/day) :

- no substance-related effects on dams , fetuses or pups

 

Thus, under the conditions of this study, Monoethanolamine pure caused some signs of maternal toxicity when administered by gavage to pregnant Wistar rats from days 6 - 15 p.c. at the highest dose level tested (450 mg/kg body weight/day). Maternal toxicity was substantiated in this dose group by a reduced food consumption, lower mean body weights and impaired body weight gain. 120 and 40 mg/kg body weight did not induce any signs of maternal toxicity in the rats.

There occurred no signs of developmental toxicity up to and including the highest dose level (450 mg/kg body weight/day). The reproductive parameters were unaffected and neither the fetuses nor the pups

showed an increased malformation rate or any indications for a substance-induced growth retardation; especially, the urinary tract of the rat fetuses/pups did not show any treatment-related findings.

Based on these study results, the no observable adverse effect level (NOAEL) on the maternal organism is 120 mg/kg body weight/day and 450 m g/kg body weight/day for the progeny.

 

 

Detailed results:

 

Maternal data:

 

Duration of Pregnancy:

The mean duration of gestation for the 21 postpartum (p.p.) females was 21.8 days, 21.6 days, 21.6 days, and 21.4 days for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. In the females euthanized on day 21 p.p., the duration of gestation and the gestation index were substantially similar in all groups.

Body weight:

The mean maternal body weight of the 450 mg/kg/day group was statistically significantly lower than that of the control group on days 15, 17, and 20 of gestation. The high dose females gained statistically significantly less weight than the controls during the treatment-free interval of the gestation period (days 15-20) and on days 0, 4, 7, and 21 p.p. The results of the corrected body weight gain on gestation day 20 of all groups did not show any differences of biological significance.

Food/water consumption:

The food consumption of the high dose animals (450 mg/kg/day) was statistically decreased within the first days of the treatment period (days 6-8 of gestation) and also after termination of the treatment on the last days of the gestation period (17-20 of gestation). During the beginning of the lactation period (days 0-4 p.p.) there was also a slight, but statistically significant reduction in the food consumption of the high dose animals, Description, severity, time of onset and duration of clinical signs: No signs that might be attributed to the test substance administered were detected during gestation and lactation periods. During gestation, piloerection was recorded for one high dose animal on day 13. Without any dose-response relationship insufficient nesting activity was observed for several dams of all groups. During lactation one dam of the 40 mg/kg/day group was found dead on day 0 p.p. after an incomplete delivery. Moreover one dam of the 120 mg/kg/day group had a total litter loss on day 1 after birth. All of these findings are spontaneous in nature and cannot be attributed to the test substance administration.

Gross pathology incidence and severity:

There were no substance-related observations at necropsy in any of the dams. Hydrometra (a spontaneous finding) was recorded for one female of the control group, for 2 females of the 40 mg/kg/day group, and 3 females of the 120 mg/kg/day group.
These animals did not become pregnant. Edema of the lungs which has to be related to the termination of the rats was recorded for several dams of the control, low and intermediate groups without any relation to dosing. For the one low dose female that died intercurrently during parturition, undelivered pups were found in the uterus.
 Organ weight changes, particularly effects on total uterine weight: The uterus weights, which were determined for the animals with termination on day 20 of gestation only, were not influenced by the administration of the test substance. The differences between the groups is without biological relevance and do not show any dose-response relationship.

 Fetal data:

Litter size and weights

Of the females euthanized on day 21 p.p. the number of females with liveborn was 12, 10, 13, and 10 and the number of pups delivered was 165, 132, 170, and 125 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. The litter size was not influenced by the test substance administration. The mean fetal body weights were not influenced by the test substance administration. The mean body weight of viable fetuses was 3.9 grams for all groups.


Number viable (number alive and number dead)

Dams with viable fetuses was 21, 20, 20 and 24 and the number of fetuses alive/dead were 293/0, 282/0, 263/0 and 311/0 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively.


Sex distribution and ratio

The sex distribution of the fetuses in the test groups was comparable with the control fetuses. Sex ratios (M/F in %) on day 0 were:

 

control: 54.6/45.4

40 mg/kg/day: 55.7/44.3

120 mg/kg/day: 46.1/53.9

450 mg/kg/day: 53.6/46.4

Sex ratios (M/F in %) on day 21 were:

control: 54.4/45.6

40 mg/kg/day: 54.0/46.0

120 mg/kg/day: 45.2/54.8

450 mg/kg/day: 54.2/45.8

The sex distribution and sex ratios of live pups on the day of birth and on day 21 p.p. did not show any substantial difference between controls and treated test groups.


Organ weights

The mean placental weights in the test groups were not influenced by the test substance administration.

 

 

Grossly visible abnormalities, external, soft tissue and skeletal abnormalities

The only external malformation which was found was an anasarca in one high-dose fetus. This malformation is also present at a low incidence in the historical control data. The external examination of the fetuses revealed no variations in any group. One unclassifed observation, fused placentae, was recorded in one fetus of the 40 mg/kg/day group and one fetus of the 450 mg/kg/day group. In all groups, including the control, some soft tissue malformations were found. These malformations were related to the eyes (microphthalmia), the heart (dilatation of the right or both ventricles; dextrocardia), the lung (uni-lobular) or the kidneys (hyper-/hypoplasia) and did not show any relation to dose. Two soft tissue variations, which were related to the urinary tract (dilated renal pelvis; hydroureter) occurred in all groups without any dose-response relationship and were fully within the historical control range. One unclassified observation (bloody inhibition of the kidneys) was recorded for 3 control and one high dose fetus.


Mortality and day of death

One dam of the 40 mg/kg/day group died intercurrently during delivery. Undelivered pups were found in the uterus.

 

Number pregnant per dose level

The conception rate varied between 85% (450 mg/kg/day group) and 75% (40 mg/kg/day group). The conception per dose level was 33 in the control group, 30 in the 40 mg/kg/day group, 33 in the 120 mg/kg/day group and 34 in the 450 mg/kg/day group. The number pregnant at caesarian-section was 21 in the control, 20 in the 40 and 120 mg/kg/day groups and 24 in the 450 mg/kg/day group. The females euthanized on day 21 p.p. showed no substance-associated effects on the fertility index which was 80%, 67%, 87% and 67% for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively.


Number aborting:

No fetuses were aborted or delivered early in any of the groups.


Number resorptions, early/late if available

Mean early resorptions were 1.4, 0.9, 1.0 and 0.9 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. Mean late resorptions were 0.2, 0.3, 0.1 and 0.0 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. In the animals euthanized on day 20 of gestation, there were no-substance related and/or statistically significant differences in the number of resorptions.


Number of implantations

The mean number of implantation sites for the 20 females euthanized on day 20 of gestation were 15.6, 15.3, 14.3 and 13.8 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. The mean number of implantation sites for the 21 p.p. females were 14.6, 14.6, 14.1 and 13.4 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. In the animals euthanized on day 20 of gestation and the females euthanized on day 21 p.p., there were no-substance related and/or statistically significant differences in the mean number of implantation sites.


Pre and post implantation loss

The mean % pre-implantation loss was 3.4, 6.8, 9.9 and 11.7 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. The mean % post-implantation loss for the 20 gestational females was 10.3, 7.3, 7.0 and 6.3 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. The mean % post-implantation loss for the 21 p.p. females was 5.7, 3.9, 9.7 and 7.3 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. In the animals euthanized on day 20 of gestation, there were no-substance related and/or statistically significant differences in the values calculated for the pre- and postimplantation losses. The females euthanized on day 21 p.p. showed no substance-associated effects on the postimplantation loss.


Number of corpora lutea

The mean numbers of corpora lutea were 16.1, 16.3, 15.6 and 15.7 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. In the animals euthanized on day 20 of gestation, there were no-substance related and/or statistically significant differences in the mean number of corpora lutea.

Postnatal growth

Without any clear relation to dosing, pup weights were occasionally statistically significantly lower in the substance-treated groups than in the respective control values. On day 21 p.p. control and high dose pup weights were substantially similar, whereas the mean pup body weights of the 40 and 120 mg/kg/day group were still slightly, but not significantly lower than the control values. On days 1-4 p.p. pup body
weight gains were also statistically significantly lower in the substance-treated groups, again without a clear dose-response relationship. Because treatment of the dams took place only until day 15 of gestation and because of no clear dose-response relationship, it seems very unlikely that the differences in pup body weight/body weight gain are substance-related.


Postnatal survival

The mean number of delivered pups/dam was not influenced by the administration of test substance. There were no substantial biological relevant differences concerning pup viability/ mortality in any of the groups. Viability and lactation indices were unaffected.


Pup clinical observations

None of the pups of the different groups showed any clinical signs until termination.

Pup necropsy observations

Only a few pups showed findings at necropsy. Post mortem autolysis, incisor sloped and dilated renal pelvis (1 high-dose pup) occurred in single pups of the control, the 40 and 450 mg/kg/day groups.

The only skeletal malformations which occurred were related to the thoracic part of the vertebral column (thoracic vertebral body/bodies dumbbell-shaped (asymmetrical) or bipartite (asymmetrical)). One or both of these malformations were found in a few fetuses of each test group including the controls without any biological relevant differences. The variations elicited were related to the ribs (shortened 13th rib(s), accessory 14th rib(s), rudimentary cervical rib(s), and the sternum (sternebra(e) of irregular shape or bipartite). These variations had no clear dose-response relationship, can be found in a similar frequency in historical control data, and/or the differences between groups are without biological significance. In all groups signs of retardations (incomplete or missing ossification of vertebral bodies/arches and the sternebra(e)) were found.
 The differences between the groups, however, are not associated with the test substance administration. All of the skeletal retardations are to be found at a comparable frequency in the historical control data and most often a clear dose-response relationship is not present. The only statistically significant difference, an increased rate of total variations in the 120 mg/kg/day group, is without biological relevance because it shows no dose-dependence.

Applicant's summary and conclusion