Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Sept. 2017 - June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
GLP conforming guideline study with no limitations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
purity: 99.6% (GC)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF SE, 97949636W0
- Expiration date of the lot/batch: 12 June 2019
- Purity: 99.6 corr. area-% (GC, DB-Wax capillary)
99.8 corr. area-% (GC, CP-Sil 5 CB/MS capillary)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Solubility and stability of the test substance in the solvent/vehicle: The test substance is completely miscible with drinking water. The stability of the test substance in drinking water over a period of 7 days at room tempera-ture was demonstrated before the start of the study in a similar batch.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Time-mated Wistar rats (Crl:WI[Han]) were supplied by Charles River Laboratories, Research Models and Services, Germany GmbH,
- Age at study initiation: about 10-12 weeks
- Weight at study initiation: 139.7 – 187.3 g
- Housing: During the study period, the rats were housed individually in Polycarbonate cages (floor area about 800 cm²). Dust-free wooden bedding was used in this study.
- Diet and water: The food used was ground Kliba maintenance diet mouse/rat “GLP” meal. Food and drinking water (potable tap water in water bottles) were available ad libitum.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The animals were accommodated in fully air-conditioned rooms in which central air condi-tioning maintained a range of temperature of 20-24°C
- Humidity (%): range of relative humidity of 30-70%
- Air changes (per hr): The air exchange rate was 15 times per hour. There were no deviations from these limits during the entire study.
- Photoperiod (hrs dark / hrs light): The day/night cycle was generally 12 hours (12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h).

IN-LIFE DATES: From 20 Sep 2017 to 05 Oct 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature.
For the test substance preparations, the specific amount of test substance was weighed, topped up with drinking water in a graduated flask and intensely mixed with a magnetic stirrer until it was completely dissolved.
Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.
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 drinking water over a period of 7 days at room temperature had been verified prior to the start of the study in a similar batch. Samples of the test substance preparations were sent once (at the beginning of administration) to the analytical laboratory for verification of the concentrations. Given that the test substance was completely miscible with drinking water, solutions were considered to be homogenous without further analysis. All test samples, plus a duplicate set of reserve samples, were withdrawn by staff of the Reproduction Toxicology. All reserve samples and further samples were stored at the Laboratory Reproduction Toxicology frozen (at -20 °C) Analysis of these samples were performed in case of equivocal analytical results with the original samples or after loss of/damage to orig-inal samples after agreement by the Study Director.
Details on mating procedure:
The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”. The animals were acclimated to the laboratory conditions between start of the study (beginning of the experimental phase) and first administration (GD 6).
Duration of treatment / exposure:
The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approximately the same time in the morning. The animals of the control group were treated with the vehicle (drinking water) in the same way. The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.
Frequency of treatment:
Daily
Duration of test:
On GD 20, the females were sacrificed in a randomized order and examined macroscopically.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
25 female animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results on preceding reproductive and developmental toxicity screening study in rats accord. to OECD TG 422.
- Rationale for animal assignment (if not random): Rats are considered to be a suitable species for the detection of a developmental toxicological hazard.
- The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

Examinations

Maternal examinations:
Mortality: A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20)
Clinical symptoms: A cage-side examination was conducted at least once daily before and after treatment period (GD 0-5 and 20). During treatment period (GD 6-19) all rats were checked daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours and within 5 hours after administration.
Food consumption: The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.
Body weight data: All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results.
Corrected (net) body weight gain: Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).
Ovaries and uterine content:
Terminal examinations of the dams:
Necropsy: On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomized order.
After the dams had been sacrificed, they were necropsied and assessed for gross pathology. The uteri and the ovaries were removed and the following data were recorded:
- 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 th e 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)

After the weight of the uterus had been determined, all subsequent evaluations of the dams and the gestational parameters (except of gross pathology including organ weights) were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.
These data were used to calculate conception rate and pre- and postimplantation losses.
Fetal examinations:
Examinations of the fetuses after dissection from the uterus:
At necropsy each fetus was weighed, sexed, and external tissues and all orifices were ex-amined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus). After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.

Soft tissue examination of the fetuses:
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.

Skeletal examination of the fetuses
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually.
Statistics:
Food consumption, body weight, body weight change, corrected body weight gain, 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:
Simultaneous comparison of all dose groups with the con-trol group using the DUNNETT-test (two-sided) for the hypothesis of equal means

Female mortality, females preg-nant at terminal sacrifice, number of litters with fetal findings:
Pairwise comparison of each dose group with the control group using FISHER'S
EXACT test (one-sided) for the hypothesis of equal proportions

Proportions of fetuses with malformations, variations and/or unclassified observations in each litter:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hy-pothesis of equal medians

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Some (7 out of 25) females of the high-dose group (1000 mg/kg bw/d) showed occasional salivation during the treatment period. Salivation occurred in the respective animals only shortly, i.e. within 0-2h, after treatment and was observed on GD 7 and 11. No clinical signs or changes of general behavior were detected in any female of all test groups beyond 2 hours after treatment. The occasional salivation was most probably caused by the bad taste or smell of the test substance and was not assessed as sign of systemic toxicity. No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 100, 300 or 1000 mg/kg bw/d during the entire study period.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 100, 300 or 1000 mg/kg bw/d).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights and the average body weight gain of the low-, mid- and high-dose dams (100, 300 and 1000 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period. The corrected body weight gain of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group (bw change from day 6: 22 g, 25 g, 24 g and 25 g for controls, 100, 300 and 1000 mg/kg bw/d, respectively). Moreover, mean carcass weights of all test groups remained unaffected by the treatment (240.5 g, 235.4 g, 243.0 g and 234.6 g for controls, 100, 300 and 1000 mg/kg bw/d, respectively).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption of the high-, mid- and low-dose dams (1000, 300 and 100 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period. The statistically significantly increased food consumption value in the low- and mid-dose dams during GD 19-20 was not considered biologically relevant due to the lack of dose re-sponse relationship.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The mean gravid uterus weights of the animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
One spontaneous finding was noted in two individual females of the control group, i.e. dilated renal pelvis.
No further necropsy findings which could be attributed to the test substance were seen in any dam.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no test substance related and/or biologically relevant differences between the different test groups in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age. See table 1 for summary of data.
The statistically significantly decreased number of implantation sites in test group 1 was assessed as an incidental finding and biologically not relevant, since all mean values (mean number 11.4/10.0*/10.6/11.0 [* = p ≤ 0.05 Dunnett-test]) were within the historical control range (implantation sites mean: 10.8 [9.9 - 11.8]). In addition, there was neither an effect on pre-implantation loss, nor a relation to dose. Therefore, this finding was not assessed as treatment-related and adverse.
Solely as a consequence of the lower number of implants, a statistically significantly decreased number of live fetuses (10.9/9.4*/9.7/10.1 [* = p ≤ 0.05 Dunnett-test]), including live female fetuses (mean number 6.0/4.2**/4.9/5.4 [** = p ≤ 0.01 Dunnett-test]), was observed in test group 1. There was neither an effect on post-implantation loss nor a relation to dose. In addition, the mean value of live fetuses was within the range of the historical control data (HCD: viable fetuses, mean: 10.0 (9.3 - 11.2)). Therefore, this finding was not assessed as treatment-related and adverse.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The conception rate was 88% in the control group (0 mg/kg bw/d), 96% in the mid-dose group (300 mg/kg bw/d) and 100% in the low- and high-dose groups (100 and 1000 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study. There were no test substance related and/or biologically relevant differences between the different test groups in conception rate. See table 1.
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Remarks on result:
other: no adverse effects observed

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean placental weights were comparable between the control and test groups 1 and 2 (0, 100 and 300 mg/kg bw/d). In test group 3 (1000 mg/kg bw/d), a statistically significantly lower mean placental weight (0.44 g) of female fetuses and of all viable fetuses (0.46 g) was observed. Both mean values were within the historical control range (female fetuses - mean 0.46 g; range 0.32 - 1.10 g; all viable fetuses - mean 0.47 g (0.35 - 1.13 g) and there was no effect on fetal weights. Therefore, these decreases were not assessed as treatment-related and adverse. The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): See table 2
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
See table 1
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was comparable to the control fetuses. See table 1
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No external malformations were recorded. No external variations were recorded. One unclassified external observation, i.e. placentae fused, was recorded in one litter of the mid-dose group (300 mg/kg bw/d). This finding was not considered as treatment-related and adverse since it was not related to dose and the mean value of affected fetuses per litter was within the historical control range (affected fetuses per litter, mean 0.0, range: 0.0 – 0.4%).
See table 3.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Skeletal malformations were noted in one fetus, each, in test groups 0, 2 and 3 (0, 300 and 1000 mg/kg bw/d. No statistically significant differences between the substance-treated groups and the control were noted and no dose-response relationship was observed. The isolated finding in test groups 2 and 3 each were single cases and the mean values of affected fetuses per litter of both findings were within the range of the historical control data. Therefore, these findings were not considered to be treatment-related. See tables 7 and 8.
For all test groups, 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 skeletons and appeared without a relation to dose. The overall incidences of skeletal variations were comparable to the historical control data The increased incidences of skeletal variations were not related to the dose and they were inside the historical control range. Therefore, they are not considered as treatment-related. See table 9.
Visceral malformations:
no effects observed
Description (incidence and severity):
One fetus of test group 3 (1000 mg/kg bw/d) had two soft tissue malformations – right-sided aortic arch and abnormal lung lobation. Abnormal lung lobation can be found in the historical control data at a comparable incidence (litter incidence, mean 0.1%, range: 0.0 - 4.0%). However, these were single events in one fetus and no cluster of any of these individual malformations were seen in the other offspring of this or the other treated groups. Thus, no association to the treatment was assumed.
The total incidence of soft tissue malformations in treated animals did not differ significantly from the concurrent control group and was covered by the historical control data. See table 5.
Four soft tissue variations were detected, i.e. misaligned palatal rugae, short innominate, dilated renal pelvis and dilated ureter. The incidences of these variations were neither statistically significantly nor dose-dependently increased in the treated groups. All of them can be found in the historical control data at comparable incidences. Therefore, they were not as-sessed as treatment-related. See table 6.
Other effects:
no effects observed
Description (incidence and severity):
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the ribs and the sternum and did not show any relation to dosing. See table 11.
Details on embryotoxic / teratogenic effects:
Assessment of all fetal external, soft tissue and skeletal observations

External malformations did not occur in any of the fetuses in this study. There were noted soft tissue and skeletal malformations in test groups 0, 2 and 3 (0, 300 and 1000 mg/kg bw/d). Two fetuses frome one litter carried more than one malformation. Female high-dose fetus No. 81-02 (1000 mg/kg bw/d) had a right-sided aortic arch and abnormal lung lobation. Female high-dose fetus No. 81-03 of the same litter had multiple malformations affecting the vertebral column, for example fused cervical arch, misshapen thoracic vertebra and absent lumbar vertebra. Further malformations, i.e. malpositioned and bipartite sternebra and cleft sternum were observed in individual fetuses, unrelated to the dose. All these findings were single cases, all of them can (except of right-sided aortic arch) be found in the historical control data. No ontogenetic pattern is recognizable for all these individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups. They also do neither form a pattern or syndrome with other minor anomalies which may raise toxicological concern nor do they influ-ence the overall rate of malformations in this study. There is no evidence for any association of these scattered findings with the treatment. See table 12

External variations did not occur in any of the fetuses in this study. Four soft tissue variations and a range of skeletal variations were noted in all test groups including the controls. None of the total incidences showed a relation to dose. The individual variations were equally distributed about the different test groups, if normal biological variation is taken into account, and can be found in the historical control data at a comparable frequency. See table 13.

No unclassified soft tissue observations were recorded for any of the fetuses in this study. A spontaneous origin is assumed for the unclassified external and unclassified skeletal cartilage observations which were observed in several fetuses of all test groups (0, 100, 300 and 1000 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment. See tables 3 and 11.

Finally, fetal examinations revealed that there is no effect of the compound on the respective morphological structures up to the highest dose tested (1000 mg/kg bw/d).



Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1

Summary of reproduction data

 

0 mg/kg bw/d

100 mg/kg bw/d

300 mg/kg bw/d

1000 mg/kg bw/d

Females mated (N)

25

25

25

25

Pregnant (N)

Conception rate (%)

22

88

25

100

24

96

25

100

Aborted (N)

0

0

0

0

Premature births (N)

0

0

0

0

Dams with viable fetuses (N)

22

25

24

25

Dams with all resorptions (N)

0

0

0

0

Female mortality (N)

0

0

0

0

Corpora lutea

Mean

SD

Total

 

11.8 D

1.63

259

 

10.9

1.89

273

 

11.5

1.67

277

 

11.7

1.44

292

Implantation sites

Mean

SD

Total

 

11.4 D

1.68

250

 

10.0 *

1.68

250

 

10.6

2.24

254

 

11.0

1.17

276

Preimplantation loss

Mean%

SD

 

3.5 D

5.66

 

7.7

10.45

 

9.4

12.90

 

5.0

7.26

Postimplantation loss

Mean%

SD

 

4.1 D

5.45

 

6.3

10.57

 

9.1

13.21

 

8.0

10.56

Resorptions Total

Mean

SD

Total

Mean%

SD

 

Resorptions Early

Mean

SD

Total

Mean%

SD

 

Resorptions Late

Mean

SD

Total

Mean%

SD

 

0.5 D

0.67

11

4.1 D

5.45

 

 

0.5 D

0.67

5.45

4.1

5.45

 

 

0.0 D

0.0

0

0.0 D

0.0

 

0.6

1.19

16

6.3

10.57

 

 

0.5

0.92

12

4.8

8.91

 

 

0.2

0.47

4

1.5

4.19

 

 

0.9

0.99

21

9.1

13.21

 

 

0.8

0.9

18

6.6

7.67

 

 

0.1

0.61

3

2.5

12.25

 

0.9

1.26

23

8.0

10.56

 

 

0.8

1.04

20

7.0

8.63

 

 

0.1

0.44

3

1.1

3.94

 

Dead fetuses (N)

0

0

0

0

Dams with viable fetuses

22

25

24

25

Live fetuses

Mean

SD

Total

 

10.9 D

1.55

239

95.9 D

5.45

 

9.4*

1.85

234

93.7

10.57

 

9.7

2.40

233

90.9

13.21

 

 

10.1

1.39

253

92.0

10.56

Females

Mean

SD

Total

Mean %

SD

 

6.0 D

2.07

132

52.7

16.04

 

4.2 **

1.42

106

42.8

13.29

 

4.9

2.21

118

45.7

15.26

 

5.4

1.50

134

49.0

14.11

Males

Mean

SD

Total

Mean %

 

4.9 D

1.83

107

43.1 D

15.89

 

5.1

2.11

128

50.9

17.24

 

4.8

1.93

115

45.2

18.45

 

4.8

1.54

119

43.0

12.65

 

Per cent live females

55.2

45.3

50.6

53.0

Per cent live males

44.8

54.7

49.4

47.0

Statistics: D : Dunnett-test (two-sided), * : p <= 0.05, ** : p < = 0.01

Table 2

Mean Fetal body weights

 

Fetal weights (g)

0 mg/kg bw/d

 

100 mg/kg bw/d

300 mg/kg bw/d

1000 mg/kg bw/d

All viable fetuses

Mean

SD

N

 

3.5 D

0.29

22

 

3.7

0.23

25

 

 

3.5

0.25

24

 

 

3.5

0.25

25

 

Male fetuses

Mean

SD

N

 

 

3.7 D

0.26

22

 

3.7

0.25

25

 

 

3.7

0.27

23

 

3.6

0.26

25

Female fetuses

Mean

SD

N

 

3.5

0.31

22

 

 

3.6

0.23

25

 

3.5

0.25

24

 

3.4

0.22

25

 

D: Dunnett-test (two-sided), * : p <= 0.05, ** : P <= 0.01

Table 3

Total external unclassified observations

 

 

 

Test group 0 0 mg/kg bw/d

Test group 1 100 mg/kg bw/d

Test group 2 300 mg/kg bw/d

Test group 3 1000 mg/kg bw/d

Litter

Fetuses

N
N

22
239

25
234

24
233

25
253

 Fetal incidence

 N (%)

0.0

0.0

1 (0.4)

0.0

 Litter incidence

 N (%)

0.0

0.0

1 (4.2)

0.0

Affected fetuses/litter

 Mean%

0.0

0.0

0.3

0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 4

Individual fetal soft tissue malformations

Test group

Dam No.-, Fetus No., Sex

Finding

0 (0 mg/kg bw/d)

none

 

1 (100 mg/kg bw/d)

none

 

2 (300 mg/kg bw/d)

none

 

3 (1000 mg/kg bw/d)

81-02 F

right-sided aortic arch, abnormal lung lobation

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

Table 5

Total soft tissue malformations

 

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter
Fetuses

N
N

22
114

25
111

24
109

25
121

 Fetal incidence

 N (%)

 0.0

0.0

0.0

1 (0.8)

 Litter incidence

 N (%)

0.0

0.0

0.0

1 (4.0)

Affected fetuses/litter

 Mean%

0.0

0.0

0.0

1.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 6

Total soft tissue variations

 

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter
Fetuses

N
N

22
114

25
111

24
109

25
121

Fetal incidence

 N (%)

1 (0.9)

3 (2.7)

3 (2.8)

3 (2.5)

Litter incidence

 N (%)

1 (4.5)

3 (12)

3 (13)

3 (12)

Affected fetuses/litter

 Mean%

0.9

2.8

2.7

 2.9

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 7

Individual fetal skeletal malformations

 

Test group

Dam No.-Fetus No., Sex

Finding

0 (0 mg/kg bw/d)

19-01 M

cleft sternum

1 (100 mg/kg bw/d)

none

 

2 (300 mg/kg bw/d)

54-07 M

malpositioned and bipartite sternebra

3 (1000 mg/kg bw/d)

81-03 F

severely malformed vertebral column

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

 

Table 8

Total skeletal malformations

 

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter
Fetuses

N
N

22
125

25
123

24
124

25
132

Fetal incidence

 N (%)

1 (0.8)

0.0

 1 (0.8)

1 (0.8)

Litter incidence

 N (%)

1 (4.5)

0.0

1 (4.2)

1 (4.0)

Affected fetuses/litter

 Mean%

0.8

0.0

0.8

0.8

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 9

Total fetal skeletal variations

 

 

 

Test group 0 0 mg/kg bw/d

Test group 1 100 mg/kg bw/d

Test group 2 300 mg/kg bw/d

Test group 3 1000 mg/kg bw/d

Litter
Fetuses

N
N

22
125

25
123

24
124

25
132

Fetal incidence

 N (%)

 123 (98)

121 (98)

123 (99)

127 (96)

Litter incidence

 N (%)

22 (100)

 25 (100)

24 (100)

25 (100)

Affected fetuses/litter

 

Mean%

 

98.5

 

98.0

 

99.0

 

96.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 10

Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter). All incidences were expressed on a fetus per litter basis.

 

Finding

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

HCD

Mean % (range)

Incomplete ossification of skull;
unchanged cartilage

3.9

8.1

18.0*

7.5

8.1

(0.8 – 21.4)

Supernumerary thoracic vertebra

2.4

2.2

7.5*

0.7

4.3

(0.8 – 11.0)

Supernumerary rib (14th); cartilage present

4.9

6.4

13.7*

3.6

7.4

(1.9 – 14.7)

Wavy rib

0.8

7.5*

7.2*

4.0

5.1

(1.4 – 13.3)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p <= 0.05 (Wilcoxon-test [one-sided]), ** = p <= 0.01 (Wilcoxon-test [one-sided])

Table  11

Total unclassified cartilage observations

 

 

 

Test group 0 0 mg/kg bw/d

Test group 1 100 mg/kg bw/d

Test group 2 300 mg/kg bw/d

Test group 3 1000 mg/kg bw/d

Litter
Fetuses

N
N

22
125

25
123

24
124

25
132

Fetal incidence

 N (%)

99 (79)

108 (88)

97 (78)

101 (77)

Litter incidence

 N (%)

 22 (100)

 24 (96)

24 (100)

24 (96)

Affected fetuses/litter

 Mean%

79.4

85.7

79.4

76.7

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 12

Total fetal malformations

 

 

 

Test group 0 0 mg/kg bw/d

Test group 1 100 mg/kg bw/d

Test group 2 300 mg/kg bw/d

Test group 3 1000 mg/kg bw/d

Litter
Fetuses

N
N

22
239

25
234

24
233

25
253

Fetal incidence

 N (%)

1 (0.4)

0.0

1 (0.4)

2 (0.8)

Litter incidence

N (%)

 1 (4.5)

0.0

1 (4.2)

 1 (4.0)

Affected fetuses/litter

 Mean%

0.4

0.0

0.4

 0.9

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 13

Total fetal variations

 

 

 

Test group 0 0 mg/kg bw/d

Test group 1 100 mg/kg bw/d

Test group 2 300 mg/kg bw/d

Test group 3 1000 mg/kg bw/d

Litter
Fetuses

N
N

22
239

25
234

24
233

25
253

Fetal incidence

N (%)

 124 (52)

124 (53)

126 (54)

130 (51)

Litter incidence

 N (%)

 22 (100)

25 (100)

24 (100)

25 (100)

Affected fetuses/litter

 Mean%

52.1

52.9

53.9

51.6

mg/kg bw/d = milligram per kilogram body weight per day; N= number; % = per cent

Applicant's summary and conclusion

Conclusions:
Under the conditions of a prenatal developmental toxicity study conducted according to OECD TG 414, the oral administration of N-(2-Hydroxyethyl)-2 -pyrrolidonto pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/day caused neither evidence of maternal nor developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 1000 mg/kg bw/day.
Executive summary:

N-(2-Hydroxyethyl)-2-pyrrolidon was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 100, 300 and 1000 mg/kg bw/day on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (drinking water) in parallel. At terminal sacrifice on GD 20, 22-25 females per group had implantation sites. 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 GD 20, all surviving females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings. The stability of the test substance preparations over a period of 7 days at room temperature was demonstrated. The correctness of the prepared concentrations was shown.

No test substance-related adverse effects on dams, gestational parameters or fetuses were observed at any dose level (0, 100, 300, 1000 mg/kg bw/day). Under the conditions of this prenatal developmental toxicity study, the oral administration of N-(2-Hydroxyethyl)-2 -pyrrolidonto pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/day caused neither evidence of maternal nor developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 1000 mg/kg bw/day.