Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 Jan 2001
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature / under light exclusion
- Stability under test conditions: The stability of the test substance preparations over a period of 7 days in a refrigerator was demonstrated.

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH / Charles River Laboratories, France
- Age at study initiation: 17-19 weeks of age
- Weight at study initiation: 2974 – 4515 g on GD 0
- Fasting period before study: no
- Housing: singly in Type 4X03B700CP cages (floor space 4264 mm2, internal height 450 mm)
- Diet: ad libitum; pelleted Kliba maintenance diet rabbit and guinea pig “GLP”, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland.
- Water: ad libitum; potable tap water in water bottles
- Acclimation period: min 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±2
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% Carboxymethylcellulose suspension in drinking water (with 0.5 mg Tween 80/100 mL)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0.2, 0.6 and 2 g/100 ml for 20, 60 and 200 mg/kg bw/d dose group respectively
- Amount of vehicle (if gavage): 10 ml/kg
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- Impregnation procedure: artificial insemination
A synthetic hormone (0.2 mL), which stimulates release of LH and FSH from the anterior pituitary lobe (Receptal®) was injected intramuscularly to the female rabbits about 1 hour be-fore insemination. The ejaculate samples used for the artificial insemination were obtained from male New Zealand White rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor as documented in the raw data. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.
Duration of treatment / exposure:
GD 6-28
Frequency of treatment:
daily
Duration of test:
GD 0-28
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-29).


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During the administration period (GD 6-28) all animals were checked daily for any abnormal clinical signs before the administration as well as within 5 hours after the administration.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated based on the obtained results. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: gross pathology, uteri and the ovaries


- Weight of the unopened uterus

- 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)


Only pregnant does were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant does with scheduled sacrifice (GD 29) were used for the calculation of mean gravid uterine weights, mean organ weights, corrected (net) body weight gain and summary of reproduction data.

The following females were excluded from the above-mentioned calculations:

Test group 0 (0 mg/kg bw/d):
• 6 females - not pregnant
• 1 female - sacrificed after abortion

Test group 1 (20 mg/kg bw/d):
• 1 female - not pregnant

Test group 2 (60 mg/kg bw/d):
• 2 females - not pregnant

Test group 3 (200 mg/kg bw/d):
• 2 females - died intercurrently
• 1 female - not pregnant
• 1 female - sacrificed after abortion
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
- Number of llive dead fetuses: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Head examinations: Yes: [ approx. half per litter and any fetus which revealed severe findings during the external examination]
- Skeletal examinations: Yes: [all per litter with and without skulls]

At necropsy each fetus was weighed and examined macroscopically for external findings. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. Individual placental weights were recorded.
Statistics:
DUNNETT-test (two-sided): Food consumption, body weight, body weight change, 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, proportions 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
FISHER'S EXACT test (one-sided): Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings
WILCOXON-test (one-sided): Proportions of fetuses with mal-formations, variations and/or un-classified observations in each litter
Indices:
The conception rate (in %) was calculated according to the following formula:
number of pregnant animals / number of fertilized animals x 100

The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
number of corpora lutea – number of implantations / number of corpora lutea x 100

The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
number of implantations – number of live fetuses /number of implantations x 100



Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Mortality:
mortality observed, treatment-related
Description (incidence):
Test group 200 mg/kg bw/d:
Mortality in two does
Test group 60 mg/kg bw/d:
No test substance-related adverse effects on does, gestational parameters or fetuses.
Test group 20 mg/kg bw/d:
No test substance-related adverse effects on does, gestational parameters or fetuses.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test group 200 mg/kg bw/d:
Corrected (net) body weight loss in individual does up to 4 times higher than the group average
Mean body weights and the average body weight gain were generally comparable to the concurrent control group throughout the entire study period.
Mean carcass weights and the corrected body weight change (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were not significantly different.
Test group 60 mg/kg bw/d:
No test substance-related adverse effects on does, gestational parameters or fetuses.
Test group 20 mg/kg bw/d:
No test substance-related adverse effects on does, gestational parameters or fetuses.

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test group 200 mg/kg bw/d:
Reduced average food consumption (10% throughout treatment period, statistically non-significant). FC was constantly below control during the treatment period, but the difference gained statistical significance only on GD 7-8.
Test group 60 mg/kg bw/d:
No test substance-related adverse effects on does, gestational parameters or fetuses.
Test group 20 mg/kg bw/d:
No test substance-related adverse effects on does, gestational parameters or fetuses.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Severe confluent reddening of the stomach mucosa in 1 high-dose doe (200 mg/kg bw/d - found dead on GD 28).
Multiple ulcerations in stomach in 1 high-dose doe (200 mg/kg bw/d - sacrificed after abortion on GD 26).
Details on results:
One female, each, of the 20 mg/kg bw/d and 60 mg/kg bw/d dose group had blood in bedding on GD 21-22, respectively.
In total, reduced defecation was observed in 2 control, 3 of the 20 mg/kg bw/d , 2 of the 60 mg/kg bw/d and 3 of the 200 mg/kg bw/d females. No defecation was observed in one female of 60, 200 mg/kg bw/d test groups, each. Incidence and distribution of these findings do not indicate a relationship to the test substance. There were no clinical findings in the other does in the study.

The mean gravid uterus weights of the rabbits of test groups 20, 60 and 200 mg/kg bw/d were not influenced by the test substance. The differences between these groups and the control group showed no dose-dependency and were assessed to be without biological relevance.

Maternal developmental toxicity

Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
Test group 200 mg/kg bw/d:
Abortion in one doe
Test group 60 mg/kg bw/d:
No test substance-related adverse effects on does, gestational parameters or fetuses.
Test group 20 mg/kg bw/d:
No test substance-related adverse effects on does, gestational parameters or fetuses.
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
Test group 200 mg/kg bw/d:
4 dead fetuses clustered in one litter; doe above-average affected by maternal toxicity, fetal findings directly linked to maternal toxicity
Test group 60 mg/kg bw/d:
No test substance-related adverse effects on does, gestational parameters or fetuses.
Test group 20 mg/kg bw/d:
No test substance-related adverse effects on does, gestational parameters or fetuses.
Details on maternal toxic effects:
There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, mean number of corpora lutea, implantation sites and resorptions, as well as the values of pre- and the postimplantation losses derived thereof.
The average number of viable fetuses was also comparable between all groups.

The conception rate was 18/ 24/ 23/ 24 (72%, 96%, 92%, 96%) in the 0, 20, 60 and 200 mg/kg bw/d respectively. A sufficient number (approximately 20, but not fewer than 16 females with implantation sites) of pregnant females was available for the purpose of the study (according to test guidelines).

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
60 mg/kg bw/day (nominal)
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
dead fetuses

Results (fetuses)

External malformations:
effects observed, treatment-related
Description (incidence and severity):
The distribution of the findings about the dose groups does not indicate an association to the treatment. No statistically significant differences between the groups were noted.
Fetal incidence: 1/0/1/6 (0.7%, 0%,0.5%, 3.5%) in the 0, 20, 60 and 200 mg/kg bw/d respectively.
Litter incidence: 1/0/1/2 (6.3%, 0%,4.3%, 9.5%) in the 0, 20, 60 and 200 mg/kg bw/d respectively.
Individual fetal external malformations included 5 cases of malrotated limbs clustered in one litter in the 200 mg/kg bw/d dose group; doe above-average affected by maternal toxicity, fetal findings directly linked to maternal toxicity.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were detected in all test groups including the control. No statistically significant differences between the test substance-treated groups and the control were noted and no dose-response relation-ship was observed.
Fetal incidence: 1/2/1/1 (0.7%, 1.1%, 0.5%, 0.6%) in the 0, 20, 60 and 200 mg/kg bw/d respectively.
Litter incidence: 1/2/1/1 (6.3%, 8.3%,4.3%, 4.8%) in the 0, 20, 60 and 200 mg/kg bw/d respectively.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Some soft tissue malformations were recorded for single fetuses of all test groups including the control. The distribution of the findings about the dose groups does not indicate an association to the treatment. No statistically significant differences between the groups were noted.
Fetal incidence: 2/2/3/2 (1.5%, 1.1%, 1.5%, 1.2%) in the 0, 20, 60 and 200 mg/kg bw/d respectively.
Litter incidence: 1/2/3/2 (6.3%, 8.3%, 13%, 9.5%) in the 0, 20, 60 and 200 mg/kg bw/d respectively.
Details on embryotoxic / teratogenic effects:
The sex distribution of the fetuses in test groups was comparable to the control fetuses. Any observable differences were without biological relevance.
The mean placental weights in test groups were not influenced by the test substance administration and were similar to the control value.
The mean fetal weights were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

External variations:
Two external variations, i.e. 5 cases of paw hyperflexion and 1 case of limb hyperextension, were recorded in test group 200 mg/kg bw/d. Three of the 5 cases of paw hyperflexion were observed in dead fetuses of one doe. These may well have been postmortal artifacts. One other case of paw hyperflexion and the case of limb hyperextension were part of a variety of limb findings (malrotated limb) in the offspring of 1 doe. No skeletal findings were evident in the extremities of any of those offspring which could be related to the defective paw positionings. Paw hyperflexions are common variants in rabbit fetuses. If the cases in dead offspring were disregarded the incidence in the present study is well covered by the historical control range.

Soft tissue variations:
The examinations of the organs revealed a broad variety of soft tissue variations, i.e. cystic dilatation of the brain, malpositioned carotid branch, narrowed pulmonary trunk, dilated aorta, enlarged ventricular chamber of the heart, absent lung lobe (lobus inferior medialis) and abnormal liver lobation, in fetuses of all test groups including the control. The incidences of these variations were neither statistically significantly different from control nor dose-dependent and therefore, not considered biologically relevant. Most of them can be found in the historical control data at comparable incidences.

Skeletal variations:
For the test groups including controls, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeleton and appeared in the majority of cases without a relation to dosing. Although the rate of affected fetuses per litter was statistically significantly increased in the low- and mid-dose groups (20 and 60 mg/kg bw/d), these incidences were well within the historical control range, while the control and the high-dose values were below. Thus, an association to the treatment is not assumed for these apparent increases.
Fetal incidence: 125/ 171/ 192/ 157 (93%, 96%, 98%, 91%) in the 0, 20, 60 and 200 mg/kg bw/d group respectively.
Litter incidence: 16/ 24/ 23 /21 (100%, 100%, 100%, 100%) in the 0, 20, 60 and 200 mg/kg bw/d group respectively.
Affected fetuses/ litter: 88.8%, 96.5%*, 98.3%**, 90.6% in the 0, 20, 60 and 200 mg/kg bw/d group respectively.
*=p<=0.05; **=p<=0.01; Wilcoxon-test [one-sided]

Skeletal variations with a statistically significant difference versus control group:
Supernumerary rib (13th); cartilage present (mean % of affected fetuses/litter): 50/ 72.4*/ 69.4*/ 57.9 in the 0, 20, 60 and 200 mg/kg bw/d group respectively (HCD 63.1 mean%; 51-69.8 range).
Incomplete ossification of talus; cartilage present (mean % of affected fetuses/litter): 0/ 3.1/ 2.5*/ 1.7 in the 0, 20, 60 and 200 mg/kg bw/d group respectively (HCD 1.1 mean%; 0-2.2 range).
*=p<=0.05; Wilcoxon-test [one-sided]

The increased incidences of skeletal variations were either not related to dose or they were inside the historical control range. Therefore, these minor changes are not considered as treatment-related adverse events.
A spontaneous origin is assumed for the soft tissue variations and the broad range of skeletal variations which were observed in fetuses of all test groups including the controls. Although the total incidence of skeletal variations was statistically significantly increased in the low- and mid-dose groups (20 and 60 mg/kg bw/d), these incidences were well within the historical control range, while the control and the high-dose values were below. Generally, all skeletal variations are equally distributed about the different test groups including controls, if normal biological variation is taken into account. The changes which were significantly different from the concurrent control were either not related to dose or can be found in the historical control data at a comparable or higher frequency.

External unclassified observations:
Two unclassified external observations, i.e. white discolored amniotic fluid and pale discolored placentae, were recorded in 3 individual litters of the high-dose group (200 mg/kg bw/d). These are considered to be indicative of the distinct maternal toxicity which was noted in the high-dose group.

Soft tissue unclassified observations:
Two unclassified soft tissue observations were recorded: a discolored thymus in one control and one high-dose fetus (200 mg/kg bw/d), and a discolored liver in one high-dose fetus (200 mg/kg bw/d). The incidence of these unclassified soft tissue observations was neither statistically significantly different from control nor dose-dependent and therefore not considered biologically relevant. A spontaneous origin is assumed. The distribution and type of these findings do not suggest any relation to treatment.

Skeletal unclassified observations:
Some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in test groups including controls. The observed unclassified cartilage findings did not show any relation to dosing and were considered to be spontaneous in nature. A spontaneous origin is assumed. The distribution and type of these findings do not suggest any relation to treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
prenatal developmental toxicity
Effect level:
60 mg/kg bw/day (nominal)
Sex:
not specified
Basis for effect level:
external malformations

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

Control low dose mid dose high dose
  20 mg/kg bw/d 60 mg/kg bw/d 200 mg/kg bw/d
Female mortality N 1 0 0 3
Nr. of pregnant / non-pregnant dams N 18 / 7 24 / 1 23 / 2 24 / 1
Nr. of dams with abortions N 1 0 0 1
Nr. of dams with early deliveries N 0 0 0 0
Nr. of dams with all resorptions N 1 0 0 0
Nr. of dams with viable foetuses  N 16 24 23 21
Resorptions Mean / Total 0.9 / 15 0.9 / 21 0.7 / 15 0.7 / 15
Preimplantation loss Mean % (SD) 9.2 (18.76) 11.8 (20.48) 5.7 (15.87) 8.1 (13.70)
Postimplantation loss Mean % (SD) 14.3 (25.11) 9.2 (12.24) 7.0 (12.71) 9.9 (15.42)
Food consumption (days 0 to 29) Mean [g/animal/d] (SD) 146.1 (24.05) 147.6 (23.32) 144.2 (27.27) 137.3 (27.00)
Body weight change (days 0 to 29) Mean [g] (SD) 561.9 (219.36) 532.4 (227.72) 474.3 (213.06) 432.5 (169.47)
Net weight change from Day 6 Mean [g] (SD) -67.2 (205.13) -93.8 (187.20) -141.2 (238.62) -192.9 (202.60)
Gravid uterine weight Mean [g] (SD) 451.8 (161.59) 437.6 (111.11) 438.7 (105.55) 456.5 (77.79)
Live fetuses Mean N / Mean % 8.4 / 91.1 7.4 / 90.8 8.5 / 93.0 8.0 / 90.1
Fetal weights male (on a litter basis) Mean [g] (SD) 38.7 (6.24) 39.4 (5.54) 36.4 (4.48) 38.7 (5.74)
Fetal weights female (on a litter basis) Mean [g] (SD) 40.0 (6.72) 40.0 (5.73) 36.7 (6.97) 37.4 (5.02)
Fetal weights combined (on a litter basis) Mean [g] (SD) 39.5 (6.25) 40.1 (5.20) 36.7 (6.23) 38.0 (5.22)
Total malformations, Fetal incidence N / % 4 / 3.0 4 / 2.2 5 / 2.6 8 / 4.6
Total malformations, Litter incidence N / % 3 / 19 3 / 13 4 / 17 4 / 19
Total variations, Fetal incidence N / % 125 / 93 171 / 96 192 / 98 157 / 91
Total variations, Litter incidence N / % 16 / 100 24 / 100 23 / 100 21 / 100

Applicant's summary and conclusion

Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of Citral to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of maternal toxicity at the high dose of 200 mg/kg bw/d, such as reduced food consumption, distinct body weight loss, doe mortality, and abortion in the most sensitive individuals. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 60 mg/kg bw/d.
Adverse fetal findings at 200 mg/kg bw/d such as mortality or limb malrotations noted in one individual litter, repectively, were a direct consequence of the maternal toxicity. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 60 mg/kg bw/d.
There is no evidence for selective developmental toxicity of Citral. The test substance is not teratogenic in rabbits at the tested dose levels.
Executive summary:

The test substance was administered to pregnant New Zealand White rabbits daily by stomach tube from implantation to one day prior to the expected day of parturition (GD 6-28) at dose levels of 20, 60 and 200 mg/kg bw/day.

Generally, no toxicologically relevant signs of maternal toxicity were observed in any of the control and test groups receiving 20 or 60 mg/kg bw/d. Neither clinical examinations nor determination of food consumption and body weights/body weight gain and necropsy revealed any relevant effect on the animals at these dose levels. However, signs of distinct maternal toxicity were noted in the high-dose group (200 mg/kg bw/d).

·        Two high-dose females were found dead during the last third of the administration period (on GD 23 and GD 28). Gross pathological examination revealed a severe confluent reddening of the stomach mucosa in one of these animals.

·        One high-dose female was sacrificed after abortion ahead of schedule (GD 26). As multiple ulcerations were found in the stomach of this doe at necropsy, a relationship of this abortion to the treatment is assumed.

·        One high-dose doe had 4 dead fetuses at term. Alike abortions, this is considered an expression of maternal toxicity in rabbits, in particular as this animal also suffered from markedly reduced food consumption (5.5 g/animal /d vs. 101.8 g/animal /d mean of the respective dose group at GD 28-29) and body weight loss (-150g at GD 28-29) near term.

·        One high dose doe with a litter including 5 cases of malrotated limbs was particularly affected by maternal toxicity in terms of constantly and markedly reduced food consumption until it almost stopped eating. Accordingly, this doe lost more (net) body weight during the treatment period than the average (net) weight loss in the high-dose group.

 

The food consumption of the high-dose group (200 mg/kg bw/d) was constantly below control during the treatment period, but the difference gained statistical significance only on GD 7-8. However, throughout treatment period (GD 6-28), the average food consumption of the high-dose does was almost 10% below the control group. Average carcass weights as well as gross and corrected (net) body weight change were also not significantly different from control in all treatment groups, although some high-dose individuals suffered from more pronounced reductions of food consumption and (net) body weight losses than the majority of animals.

Generally, these findings are indicative of a local irritating potential of the test item in the gastrointestinal tract which because of the peculiarity of rabbit digestive system subsequently led to reduced food consumption, distinct body weight loss, doe mortality, abortion and fetal mortality in the most sensitive individuals exposed to the top dose of 200 mg/kg bw/d. It is notable that all casualties occurred towards the end of pregnancy, when the rapid growth of the offspring makes it a particularly demanding pregnancy phase to the mothers.

 

There were no differences of biological relevance between the control and the substance-treated groups (20, 60 and 200 mg/kg body weight/day) in conception rate, mean number of corpora lutea, total implantations, resorptions and live fetuses, fetal sex ratio or in the values calculated for the pre- and the postimplantation losses.

 

No test substance-related differences were recorded for placental and fetal body weights, or for fetal sex ratio.

 

With one exception the external, soft tissue and skeletal examinations of the fetuses revealed no differences between the controls and the test substance-treated groups, which might be related to the test substance. Number and type of fetal external, soft tissue and skeletal findings, which were classified as malformations and/or variations, did not show any differences of toxicological relevance between the groups.

 

The exception are five cases of malrotated limbs which were clustered in one litter of the high-dose group. These findings were accompanied by pale discolored placentae in some offspring of this litter. Malrotation of the limbs is occasionally also observed in untreated control fetuses. Generally it is seen as a subtle inward rotation of the limb and in many cases is a result of reduced amnion levels and/or compression of the uterus caused by maternal toxicity, combined with some influence of positioning of the foetus in the uterus. This doe was particularly affected by maternal toxicity. It constantly and markedly reduced its food consumption from GD 18 (61% of the average in the high-dose group) onwards until it almost stopped eating from GD 27 onwards. Accordingly, this doe lost 766.5 g (net) body weight during the treatment period while the average (net) weight loss in the high-dose group was 192.9 g. As proven by skeletal examination the limb malrotations were not caused by any abnormalities of the underlying skeleton. Considering all this, there is sufficient evidence that these fetal findings are a direct consequence of the severe maternal toxicity. No similar or less severe findings of related nature were noted in any doe of the high-dose group less affected by maternal toxicity.

Altogether there is no evidence for selective developmental toxicity of the test substance. The test substance is not teratogenic in rabbits at the tested dose levels.