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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From May 07, 2003 to April 22, 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed according to OECD test guideline No. 414 and in compliance with GLP. The study was fully reliable (Klimisch score = 1), however the reliability score was lowered to 2 which is the maximum score for read-across. The supporting substance is considered adequate for read-across purpose (see Iuclid section 13 for additional justification).
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected on May 15 to June 26, 2001 / signed on September 24, 2001
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): 02/0449-1
- Physical state: Liquid / colourless - yellowish
- Homogeneity: Homogeneous
- Storage condition of test material: Refrigerator, protected from light
- Stability: Stability under storage conditions was confirmed by reanalysis

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: 70-84 days
- Weight at study initiation: Pregnant animals body weight on day 0: 148-183.6 g
- Housing: Animals were housed singly from day 0- 20 p .c. in type DK III stainless steel wire mesh cages
- Diet: Ground Kliba maintenance diet rat/mouse/hamster meal (PROVIMI KLIBA SA, Kaiseraugst, Switzerland), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 6 days

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

IN-LIFE DATES: From: May 07, 2003 To: May 29, 2003

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in olive oil Ph.Eur./DAB were prepared at the beginning of the administration period and thereafter at intervals, which took into account the analytical results of the stability verification. For the preparation of the solutions, an appropriate amount of the test substance was weighed in a graduated beaker (depending on the dose group), topped up with olive oil Ph.Eur./DAB, and subsequently thoroughly mixed using a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 5, 20 and 80 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in olive oil for a period of at least 4 days in refrigerator (at 4 °C) were carried out.
Samples of the test substance solutions were analysed twice during the study period (at the beginning and towards the end) for verification of the concentrations. Since the test substance preparations were true solutions, investigations concerning homogeneity were not necessary.
Details on mating procedure:
- Impregnation procedure: Purchased timed pregnant
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post coitum (p.c.)
Duration of treatment / exposure:
Test substance was administered from implantation to one day prior to the expected day of parturition (day 6 to day 19 p.c.)
Frequency of treatment:
Once daily
Duration of test:
13 days (day 6 to day 19 p .c.)
Doses / concentrations
Remarks:
Doses / Concentrations:
25, 100 and 400 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
25 mated females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 25 mg/kg bw/day: as the expected no observed adverse effect level; 100 mg/kg bw/day: as intermediate dose level; 400 mg/kg bw/day: as the dose level which should induce some developmental and/or maternal toxicity but no death or severe suffering.
- Rationale for animal assignment: The animals were mated by the breeder and supplied on day 0 post coitum. They were assigned to the test groups by taken random selection from the transport box.

Examinations

Maternal examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Mortality was checked twice a day on working days or once a day (Saturday, Sunday or on public holidays) (days 0- 20 p.c.). The animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited (days 0- 20 p.c.).

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 p.c.
- Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on day 20 p.c. minus weight of the unopened uterus minus body weight on day 6 p.c.).

FOOD CONSUMPTION: Yes
- Time schedule for examinations: With the exception of day 0, the consumption of food was determined on the same days as was body weight.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- On day 20 p.c., the dams were sacrificed by cervical dislocation and the fetuses removed from the uterus.
- Organs examined: Uterus and the ovaries were removed and the following data were recorded: weight of the liver and, weight of the unopened uterus
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 (only decidual or placental tissues visible or according to SALEWSKI (Salewski, 1964) from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy)
- Number of late resorptions: Yes (embryonic or fetal tissue in addition to placental tissue visible)
- Other: dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)
Fetal examinations:
At necropsy each fetus was weighed, sexed and examined macroscopically for any external findings. 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. Thereafter, the fetuses were sacrificed by subcutaneous injection of a pentobarbital. After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and placed in ethyl alcohol, the other half was placed in Harrison's fluid for fixation and further evaluation .
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]; The fetuses fixed in Harrison's fluid were examined for any visceral findings according to the method of Barrow and Taylor (1969).
- Skeletal examinations: Yes: [half per litter]; The skeletons of the fetuses fixed in ethyl alcohol were stained according to a modified method of Kimmel and Trammell (1981). Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually.
Statistics:
See Table 7.8.2/1
Indices:
Conception rate (%) = (number of pregnant animals / number of fertilized animals) x 100
Pre-implantation loss (%) = ((number of corpora lutea - number of implantations) / number of corpora lutea) x 100
Post-implantation loss (%) = ((number of implantations - number of live fetuses) / number of implantations) x 100
Historical control data:
Historical control data (July to October 2001) were provided to compare with concurrent controls.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical examinations
Only pregnant dams were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant dams with scheduled sacrifice (day 20 p.c.) were taken for the calculation of mean gravid uterine weights, mean net maternal body weight change (corrected body weight gain) and summary of reproduction data.

Mortality:
There were no substance-related or spontaneous mortalities in any of the groups.

Clinical symptoms:
At 400 mg/kg bw/day, transient salivation in all rats immediately after gavaging on days 6 - 19 p.c. At 100 mg/kg bw/day, transient salivation in 22 out of 25 rats immediately after gavaging between treatment days 10 - 19 p.c. After cessation of treatment on day 19 p.c., salivation did not occur any longer. The observed temporary salivation of the animals is considered to be substance-induced. It is very likely, that this finding was induced by bad taste of the test substance or local affection of the upper digestive tract. Salivation itself is not assessed as an adverse or toxic effect.
At 400 mg/kg bw/day, discoloured urine in a total of 21 out of 25 dams (days 12 - 20 p.c.). This urine discoloration is probably related to a chemical reaction of the test substance or its metabolites with the bedding or with components of the air; it does not represent a toxicologically relevant finding.

Body weight:
The mean body weights of the low, mid and high dose rats (25, 100 and 400 mg/kg bw/day) were substantially similar to the concurrent control values. The observable, insignificant differences between the substance-treated groups and the controls are without any biological relevance. A statistically significant impairment in mean body weight gain (about 29% below the concurrent control value) occurred in high dose group on treatment days 8 - 10 p.c.; on the other treatment days (i.e. days 6 - 8 and 10 -19 p.c.) weight gains of the 400 mg/kg bw/day females were sometimes below and sometimes above those of the corresponding controls without attaining statistical significance. As the food consumption of these rats was also transiently diminished and the corrected body weight gain was also slightly decreased, this is considered to be a substance-related sign of maternal toxicity. Body weight gains of the dams of test groups (25 and 100 mg/kg bw/day) were similar to those of the concurrent controls.

Corrected body weight gain (net maternal body weight change):
The corrected body weight gains (terminal body weight on day 20 p.c. minus weight of the unopened uterus minus body weight on day 6 p.c.) of the dams of test groups (25 and 100 mg/kg bw/day) revealed no differences of any biological relevance to the corresponding control group. The net weight change of the 400 mg/kg bw/day rats, however, was about 17 % below the concurrent control value (without attaining statistical significance). As food consumption and body weight gain of the 400 mg/kg bw/day rats were also temporarily diminished, the effects on net body weight gain at the top dose are considered to be substance-related, borderline signs of maternal toxicity.

Food consumption:
The mean food consumption of the high dose dams (400 mg/kg bw/day) was statistically significantly reduced (9 % below the concurrent control value) at initiation of treatment (days 6 - 8 p.c.). On the following days of the treatment period, however, food consumption of the high dose rats reached or even exceeded control values. At 25 and 100 mg/kg bw/day, the food consumption of the females was unaffected and did not show any statistically significant or biologically relevant differences in comparison to the controls. The transient reductions in food consumption at 400 mg/kg bw/day were accompanied by corresponding impairments in body weight gain of these dams at initiation of dosing. Therefore, this finding is considered to be substance-induced.

Organ weights
Uterus weight: The mean gravid uterus weights of the animals of test groups (25, 100 or 400 mg/kg bw/day) were not influenced by the administration of the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance. They reflect the normal degree of variation for rats of the strain used for this study and have to be assessed in association with the fortuitous fluctuations in the mean number of live fetuses/dam in this study.
Liver weight: Absolute and relative mean liver weights were statistically significantly increased at the mid and high dose groups (100 and 400 mg/kg bw/day) and were about 9 or 29 % (absolute) and 8 or 29 % (relative) above control values. These weight increases, which are considered to be substance-induced, are indicative of hepatic changes primarily caused by microsomal enzyme induction. Absolute and relative liver weights of the dams of test group 1 (25 mg/kg bw/day), however, were similar to the respective control values and did not show any toxicologically significant changes.

Necropsy findings:
No substance-related observations at necropsy in any of the dams of test (0, 25, 100 or 400 mg/kg bw/day). Only very few spontaneous findings were recorded for single low and mid dose rats:
hydrometra in low dose female, which consequently did not become pregnant,
hemorrhagic thymus in mid dose female
No association to the test compound is assumed for these findings due to their scattered occurrence without any relation to dosing.

Reproduction data of dams
The conception rate is 96, 92, 100 and 92 % at 0, 25, 100 and 400 mg/kg bw/day, respectively. There were no substance-related and/or biologically relevant differences between the test groups in the conception rate, in the mean number of corpora lutea and implantation sites, the pre- and the post implantation losses, and the number of resorptions and viable fetuses. The pre- and the post implantation loss values in the 25 and 100 mg/kg bw/day groups, however, were above the upper ranges of the historical control values and the mean number of live fetuses/ low dose dam (25 mg/kg bw/day) was statistically significantly below the concurrent and the historical control value. These differences appeared without any dose-response relationship and thus are not considered to reflect any substance-induced effect.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Sex distribution of fetuses
The sex distribution of the fetuses at 25, 100 and 400 mg/kg bw/day was comparable with that of the control fetuses. The differences observed in comparison to the control were without any biological relevance.

Weight of placentae
The mean placental weights in the substance-treated groups (25, 100, and 400 mg/kg bw/day) did not show any differences with toxicological relevance and were generally similar to the corresponding control values. The statistically significant increase of the mean placental weight of the high dose male fetuses (0 .45* g versus 0.41g in the concurrent control group; *= p≤0.05) has no biological relevance, is not considered to demonstrate an adverse finding and the respective value is fully within the historical control range (0.32-0.58 g).

Weight of fetuses
The mean fetal body weights in test groups (25, 100 and 400 mg/kg bw/day) were not influenced by the test substance administration and were very similar to or even identical with concurrent control values.

External examination of the fetuses
External malformations were recorded for one fetus each of test groups (0 and 100 mg/kg bw/day). The isolated and scattered occurrence of external malformations in one control and one mid dose (100 mg/kg bw/day) fetus does, of course, not suggest any treatment relationship. No external variations and unclassified external observations were recorded for any of the fetuses. The mean percentages of affected fetuses/litter with external malformations amounted to 0.4, 0.0, 0 .6 and 0.0 % at 0, 25, 100 and 400 mg/kg bw/day, respectively.

Soft tissue examination of the fetuses
The only soft tissue malformation (situs inversus) occurred in one low dose fetus. None of the other examined fetuses showed visceral malformations. The isolated and scattered occurrence of one soft tissue malformation in one low dose fetus does not suggest any treatment relationship. The mean percentages of affected fetuses/litter with soft tissue malformations amounted to 0.0, 1.2, 0.0, and 0.0 % at 0, 25, 100 and 400 mg/kg bw/day, respectively.
Two soft tissue variations (uni- or bilateral dilation of the renal pelvis and/or ureter) were detected in each group including the controls. The mean percentages of affected fetuses/litter with total soft tissue variations amounted to 7.5, 6.0, 2.9 and 8.8 % at 0, 25, 100 and 400 mg/kg bw/day, respectively. Thus, a clear relation to dosing is not present if normal biological variation is taken into account (historical control range 4.0- 22.2 % affected fetuses per litter). Therefore, a substance-induced effect concerning the occurrence of soft tissue variations can be excluded with certainty. No unclassified soft tissue observation (like blood imbibition of kidney(s)) was recorded in any of the fetuses.

Skeletal examination of the fetuses
Malformations of the skeletons were observed at low incidences in fetuses at 0 and 100 mg/kg bw/day, but not observed at 25 and 400 mg/kg bw/day. The mean percentages of affected fetuses/litter with skeletal malformations amounted to 0.7, 0.0, 0.9, and 0.0 % at 0, 25, 100 and 400 mg/kg bw/day, respectively. The noted skeletal malformations appeared without any dose-response relationship, have no toxicological relevance due to their scattered occurrence and/or can be found at low, but comparable incidences in the historical control data. The mean percentages of affected fetuses/litter with skeletal variations amounted to 95.4, 93.5, 96.3 and 89.2 % at 0, 25, 100 and 400 mg/kg bw/day, respectively. These incidences do not suggest a substance-induced background. Unclassified cartilage observations related to the skull, vertebral column, ribs, and the sternum occurred in all groups including the controls. The mean percentages of affected fetuses/litter with these findings amounted to 17.0, 18.5, 15 .4 and 13.6 % at 0, 25, 100 and 400 mg/kg bw/day, respectively. A toxicological relevance for these findings, which did not show any relation to dosing, can be excluded with certainty.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

ANALYSES

Stability analysis of the test substance preparations: The stability of the test substance solutions in olive oil over a period of 4 days in the refrigerator (at 4 °C) could be demonstrated.

 

Concentration analyses of the test substance preparations: The results of the analyses of the test substance solutions in olive oil Ph.Eur./DAB confirmed the correctness of the prepared concentrations. The analytical values of the samples corresponded to the expected values within the limits of the analytical method, i.e. were always above 90 % and below 110 % of the nominal concentrations. Actually, the mean values were found to be in a range of 94.8-104.1 % of the nominal concentration.

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the NOAEL for maternal and prenatal developmental toxicity are 100 and 400 mg/kg bw/day, respectively in rats.
Executive summary:

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, test substance was administered by oral (gavage) to groups of mated female Wistar rats (25/dose) at the dose levels of 0 (olive oil), 25, 100 and 400 mg/kg bw/day from Days 6 to 19 post-coitum (p.c.). Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On day 20 post coitum, all females were sacrificed and assessed by gross pathology (including weight determinations of the liver, the unopened uterus and the placentae). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated as resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed, and further investigated for any external findings. Thereafter, nearly one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (including cartilage) findings.

 

The following substance-related findings were obtained:

 

At 400mg/kg bw/day:

- transient salivation in all rats immediately after gavaging on days 6 -19 p.c.

- discoloured urine in a total of 21 out of 25 dams(days 12 - 20 p.c.)

- statistically significantly reduced food consumption on days6 - 8 p.c. (about 9 % below controls)

- statistically significant impairments in absolute body weight gain on days 8-10 p.c. (about 29 % below controls)

- lower corrected body weight gain (about 17 % below controls) without attaining statistical significance

- statistically significantly increased absolute and relative liver weights(about 29 % above controls)

- no substance-related effects on gestational parameters or fetuses.

 

At 100mg/kg bw/day:

- transient salivation in 22 out of 25 rats immediately after gavaging between treatment days 10-19 p.c.

- statistically significantly increased absolute and relative liver weights (about 8 or 9 % above controls)

- no substance-related effects on gestational parameters or fetuses.

 

At 25 mg/kg bw/day:

- no substance-related adverse effects on dams, gestational parameters or fetuses.

 

Treatment with test substance elicited substance-induced effects on the dams including signs of maternal toxicity at 400 mg/kg bw/day. 100 mg/kg bw/day resulted in some substance-related findings (i.e. temporary salivation, marginally increased liver weights), which are, however, not considered to be adverse, but some adaptive responses of the animals. At 25 mg/kg bw/day, no substance-induced effects on the dams occurred. The test substance had no influence on gestational parameters and induced no adverse signs of developmental toxicity up to and including the high dose level (400 mg/kg bw/day); especially, no indications of teratogenic effects occurred which could be causally related to the test substance administration. 

 

Under the test conditions, the NOAEL for maternal and prenatal developmental toxicity are 100 and 400 mg/kg bw/day, respectively in rats. The supporting substance is considered adequate for read-across purpose (see Iuclid section 13 for additional justification).