Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline 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)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Remarks:
Experimental Toxicology and Ecology, BASF AG
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): lonon R
- Test substance No.: 02/0449-1
- Physical state: liquid / colorless - yellowish
- Analytical purity: 97.8% (Analytical Report: 02L00370)
- Lot/batch No.: continuous production
- Date of production: October 09, 2002
- Stability under test conditions: under storage conditions was confirmed by reanalysis (Analytical Report 03L00294)
- Storage condition of test material: Refrigerator, protected from light
- Other: Analytical laboratory: Analytical Department, BASF Aktiengesellschaft, Ludwigshafen/Rhein, Germany

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Time-mated Wistar rats (CrIGIxBrIHan:WI)
- Source: Charles River Laboratories, Germany
- Animal identification: by ear tattoo
- Age at study initiation: 70 - 84 days
- Weight at study initiation: 148 .0 - 183.6 g
- Housing: singly from day 0 - 20 p .c. in type DK III stainless steel wire mesh cages supplied by BECKER & CO., Castrop-Rauxel, FRG (height : 15 cm, length: 37,5 cm, width: 21 cm; floor area about 800 cm2)
- Diet (e.g. ad libitum): ad libitum, ground Kliba maintenance diet rat/mouse/hamster meal (PROVIMI KLIBA SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): ad libitum, drinking water of tap water quality from water bottles
- Acclimation period: 6 days

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

ANALYSES
- Food: assayed for chemical as well as for microbiological contaminants
- Water: regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by Technical Services of BASF Aktiengesellschaft as well as for the presence of microorganisms by a contract laboratory

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):prepared at the beginning of the administration period and thereafter at intervals, which took into account the analytical results of the stability verification .
- Mixing appropriate amounts with (Type of food): 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
- Lot/batch no. (if required): Ph .Eur./DAB
- Amount of vehicle (if gavage): 5 ml/kg bw olive oil
- Concentration in vehicle: 500, 2000 and 8000 mg/100 ml
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test substance solutions were sent to the analytical laboratory 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:
The animals were mated by the breeder ("time-mated") and supplied on day 0 post coitum (= detection of vaginal plug / sperm). The animals arrived  on the same day (i.e. day 0 p.c.) at the experimental laboratory. The  following day was designed "day 1" post coitum (p.c.). Animals were  assigned to the test groups by taken random selection.
Duration of treatment / exposure:
day 6 through day 19 post coitum (p.c)
Frequency of treatment:
once daily
Duration of test:
On day 20  p.c., all surviving females were sacrificed
Doses / concentrations
Remarks:
Doses / Concentrations:
25, 100, and 400 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The following doses were chosen for the present full-scale toxicity study in Wistar rats. The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes, check for mortality
- Time schedule: twice a day on working days or once a day (Saturday, Sunday or on public holidays) (days 0- 20 p .c.).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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: on days 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 p.c.
The body weight change of the animals was  calculated from these results

CORRECTED BODY WEIGHT GAIN (net maternal body weight change): Yes
- Time schedule: 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 AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: With the exception of day 0, the consumption of food  was determined on the same days as was body weight

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: liver, uterus, ovaries
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: dead fetuses

- Other: calculations of conception rate and pre- and postimplantation losses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes:half per litter per litter
- Skeletal examinations: Yes: half per litter per litter
- Head examinations: Yes: all per litter
Statistics:
- two-sided Dunetts: Food consumption, body weight, body weight changes, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportion of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight
- two-sided Kruskal-Wallis test: liver weights
one-sided Fisher's Exact test: female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings
- one-sided Wilcoxon test: proportions of fetuses with malformations, variations and/or unclassified observations in each litter
Historical control data:
The historical control data used for interpretation of findings refer to  the same test facility, the same rat strain and supplier of the animals  and cover a period of about 24 months (June 2001 - June 2003, 15 studies)

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
400 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
400 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

Result: no influence on gestational parameters, no adverse signs of developmental  toxicity, no indications of teratogenic effects


TEST SUBSTANCE ANALYSES
The stability of the test substance suspensions over a period of 4 days  (at 4°C) and the correct concentration of the test substance in the test  preparation was demonstrated (always above 90% and below 110% of nominal  concentrations). 

MATERNAL TOXIC EFFECTS BY DOSE LEVEL
- Mortality and day of death: There were no substance-related or  spontaneous mortalities in any of the groups.
- Clinical examinations: Each test group including the controls contained  a sufficient number of females with implantation sites at necropsy (20 or  more).

Clinical symptoms: All high dose and the majority (22 out of 25) of the  mid dose animals showed transient salivation immediately after treatment on one or several days of the treatment period; however, the observed  salivation persisted in the respective females only for a few minutes  after the actual gavaging had taken place. After cessation of treatment  on day 19 p.c., salivation did not occur any longer. The observed  temporary salivation of the animals was 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. 

Additionally, 21 high dose dams showed dark-yellow discolored urine on  gestation days 12 - 20 p.c. which is probably related to a chemical  reaction of the test substance or its metabolites with the bedding or  with components of the air and does not represent a toxicologically  relevant finding. Comparable urine findings have been observed in a  4-week range finding study in Wistar rats with dietary administration of  the test substance, Project No.: 30S0449/02045 (BASF AG, 2003).
No indications for disturbances of the general behavior, however,  occurred in the control and low dose dams.

- Food consumption: The mean food consumption of the high dose dams 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. The food consumption of the  females of low and mid dose dams 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 were  accompanied by corresponding impairments in body weight gain of these  dams at initiation of dosing. 
 
- Body weight data: The mean body weights of the low, mid and high dose  rats were substantially similar to the concurrent control values. 
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 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 was considered to be a substance-related  sign of maternal toxicity.
Body weight gains of the dams at 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 the low and mid dose groups revealed no differences of any  biological relevance to the corresponding control group. The net weight  change of the 400 mg/kg bw rats, however, was about 17% below the  concurrent control value (not statistically significant). As food  consumption and body weight gain of this group was 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.

EXAMINATION OF THE DAMS AT TERMINATION

- Uterus weight: The mean gravid uterus weights of the animals of all  test groups were not influenced by the administration of the test  substance.  

- Liver weight: Absolute and relative mean liver weights were  statistically significantly increased at the mid and high dose groups 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  low dose dams, however, were similar to the control values and did not  show any toxicologically significant changes.

- Necropsy findings: There occurred no substance-related observations at  necropsy in any of the dams of all test groups. Only very few spontaneous  findings were recorded for single low and mid dose rats (one hydrometra  in low dose female which consequently did not become pregnant,  hemorrhagic thymus in  one mid dose female). No association to the test  compound was assumed for these findings due to their scattered occurrence  without any relation to dosing.

- Reproduction data of dams: The conception rate reached 96% in the  controls, 92% in the low and the high dose groups, and 100% in the mid  dose. 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  postimplantation losses, and the number of resorptions and viable fetuses  (see table below). The pre- and the postimplantation loss values in the  25 and 100 mg/kg groups, however, were above the upper ranges of the  historical control values and the mean number of live fetuses/low dose  dam 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. They can well be explained by the fact that one  low dose dam and two mid dose dams resorbed all of their implants and  thus had no viable fetuses (the same was also observed for one control  dam). Moreover, one low dose, which had 7 implantation sites, resorbed 6  implants and had only one live fetus at terminal sacrifice. If these 5  rats are excluded from the calculation of the means, pre- and  postimplantation loss values as well as the mean number of live  fetuses/dam fit well into the historical control ranges with one  unimportant exception (preimplantation loss value at 25 mg/kg bw/day).

Table: Pre- and postimplantation loss (PRI and POI) values and mean  number of live fetuses per dam

Dose         Control Low    Mid   High    Historical
                            dose          control range


PRI          6.0     14.4   12.7   4.7    8.7 (3.5-12.2)
Corrected    4.8     12.9*   8.7   4.7    

POI          9.9     14.8   15.2   6.2    6.7 (3.7-11.3)
Corrected    6.0      7.3    7.9   6.2    

Live fetuses 8.7      7.4*   8.2   8.6    8.8 (7.9-9.8)
per dam                                       
Corrected    8.7      7.7    8.2   8.6

Corrected: exclusion of 5 dams,  *   P < = 0.05

EXAMINATION OF FETUSES

- Sex distribution of fetuses: The sex distribution of the fetuses in all  test groups was comparable with that of the control fetuses.
 - Weight of placentae: The mean placental weights in the  substance-treated groups did not show any differences with toxicological  relevance. The statistically significant increase of the mean placental  weight of the high dose male fetuses (0.45g versus 0.41g in the control  group; p <= 0.05) is not considered to demonstrate an adverse finding and  the respective value was fully within the historical control range  (0.32g-0.58g).
- Weight of fetuses: The mean fetal body weights in all test groups were  not influenced by the test substance administration and were very similar  to or even identical with concurrent control values.
- Fetal external, soft tissue and skeletal observations:
The scattered occurrence of the few observed external, soft tissue and  skeletal malformations in single fetuses of the controls, the low and mid  dose group without a consistent pattern, without any dose-response  relationship and/or at incidences, which are similar to historical  control rates did not suggest any substance-induced origin of these  findings. Most of the observed malformations were limited to one multiply  malformed mid dose fetus. The external examination of this fetus revealed  gastroschisis, anal atresia, malrotated left hindlimb, and a thread-like  tail. During its skeletal evaluation additional malformations like  severely malformed sternum and absent vertebrae were recorded. The other  malformations that occurred were anasarca (in one control fetus), situs  inversus (in one low dose fetus), and cleft sternum (in another control  fetus).

If all different types of malformations are summarized, in total 2 of the  200 examined control fetuses [= 1.0%] in 2 out of 23 litters [= 8.7%],  one of the 163 examined low dose fetuses [= 0.6%] in one out of 22  litters [= 4.5%], one of the 189 examined mid dose fetuses [= 0.5%] in  one out of 23 litters [= 4.3%] and none of the 197 examined high dose  fetuses (from 23 litters) showed malformations. The mean percentages of  affected fetuses/litter with total malformations amounted to 0.8, 0.5, 0.6, and 0.0% at 0; 25; 100 or 400 mg/kg bw/day respectively. These low, non dose-related incidences did not suggest any relationship to the test  substance.

External variations did not occur in any of the fetuses in this study.  Soft tissue variations, exclusively in the form of dilated renal pelvis  and/or ureters, and a broad range of skeletal variations occurred in all  test groups including the controls. All fetal and litter incidences for  these variations and the corresponding mean percentages of affected  fetuses/litter did not show a clear relation to dosing, were not  considered to be of any toxicological relevance and/or could be found at  a comparable frequency in the historical control data. This statement  included the statistically significantly increased occurrence of three  variations (i.e. supraoccipital holes, bipartite ossification of thoracic  centrum [with dumbbell-shaped cartilage of centrum] and supernumerary  14th rib [without cartilage]) at the mid dose group.

If all variations were summarized, in total 105 of the 200 examined  control fetuses [= 53%] in all 23 litters [= 100%], 88 of the 163  examined low dose fetuses [= 54%] in all 22 litters [= 100%], 99 of the  189 examined mid dose fetuses [= 52%] in all 23 litters [= 100%] and 97  of the 197 examined high dose fetuses [= 49%] in all 23 litters [= 100%]  showed variations. The mean percentages of affected fetuses/litter with  total variations amounted to 52.8, 56.2, 52.9, and 50.8% at 0; 25, 100 or  400 mg/kg bw/day respectively. These incidences did not suggest a  treatment-relationship, but reflect the usual biological variation  inherent in the strain of rats used for this experiment.

A spontaneous origin was also assumed for the few unclassified cartilage  observations which were recorded for several fetuses of all test groups.  Distribution and type of these findings do not suggest any relation to  treatment as 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 or 400  mg/kg bw/day, respectively.

The following substance-related findings were obtained:

Test group 3 (400 mg/kg body weight/day):

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

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

• statistically significantly reduced food consumption on days 6 - 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

Test group 2 (100 mg/kg body weight/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

Test group 1 (25 mg/kg body weight/day):

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

Applicant's summary and conclusion