Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-25 until 2019-03-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 Jan 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
CAS nr used in the past for this type of chemical was 85204-21-3, after analytical charaterisation renamed and new CAS 1471311-93-9 was applied.
Batch identification: 14671736W0
Purity: 100% UVCB substance
Content: 100 g/100 g (UVCB)
Content of Triethanolamine: 74.2 g/100 g (Analytical Report, study code: 17L00233)
Specific details on test material used for the study:
Please note: This kind of substance was in the past handle under the CAS nr 85204-21-3
After analytical characterisation the substance was renamed and received a new CAS nr. Now the substance is characterised by the CAS 1471311-93-9 EC Number: 939-488-3

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: BASF and 14671736W0
- Expiration date of the lot/batch: 01 May 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the vehicle: stability in drinking water with 5 mg/100 mL Cremophor EL over a period of 7 days at room temperature verified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: prepared at the beginning of the administration period and thereafter at intervals, specific amount of test substance was weighed, topped up with drinking water or deionized water
- Final dilution of a dissolved liquid: suspension in drinking water or deionized water with 5 mg/100 mL Cremophor EL, respectively

FORM AS APPLIED IN THE TEST: suspension; preparations were kept homogeneous with a magnetic stirrer

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11-13 weeks
- Weight at study initiation: 146-193 g (day of arrival = gestation day 0 (GD 0))
- Housing: housed individually in Polycarbonate cages type III, floor area about 800 cm² (TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany)
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA (new name Granovit AG), Kaiseraugst, Switzerland
- Water: ad libitum, tap water in water bottles
- Acclimation period: 6 days (GD 0 - GD 6)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (6.00 h to 18.00 h / 18.00 h to 6.00 h)

IN-LIFE DATES: From: 25 Jan 2018 To: 15 Feb 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
drinking water, from day 8 onwards deionized water (with 5 mg/100 mL Cremophor EL, respectively
Details on exposure:
APPLICATION OF DOSING SOLUTION:
- volume administered: 10 mL/kg body weight

PREPARATION OF DOSING SOLUTIONS:
- specific amount of test substance was weighed, topped up with drinking water or deionized water
- prepared in a calibrated beaker and intensely mixed with a magnetic stirrer
- Rate of preparation: at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: water with 5 mg/100 mL Cremophor EL
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- samples of the test substance preparations were analyzed (at the beginning of administration and due to a change in the vehicle) for verification of the concentrations
- samples taken for the concentrations control analyses were also used to verify the homogeneity of the samples of the low- and high-concentrations each (100 and 1000 mg/kg bw/d)
- three samples (one from the top, middle and bottom) were taken for each of these preparations from the preparation vessel with a magnetic stirrer running
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy (GD 0)
Duration of treatment / exposure:
from implantation to one day prior to the expected day of parturition (GD 6 to GD 19)
Frequency of treatment:
once a day, always at approximately the same time in the morning
Duration of test:
21 days (GD 0 until GD 20)
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
1 g/ 100 mL
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
3 g/ 100 mL
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
10 g/ 100 mL
No. of animals per sex per dose:
25 female animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: as requested by the sponsor, based on available studies e.g. 85R0586/11S125
- Rationale for animal assignment: each cohort was evenly distributed among the dose groups

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day, once on Saturdays, Sundays or on public holidays (GD 0-20)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily before and after treatment period (GD 0-5 and 20); during GD 6-19 all rats were checked daily before administration as well as within 2 hours and within 5 hours after administration
- Parameters: any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: apart from uterus, not further specifications
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: number of dead fetuses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
Statistics:
- Simultaneous comparison of all dose groups with the control group, DUNNETT-test (two-sided) for the hypothesis of equal means: 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

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

- Pairwise comparison of each dose group with the control group, WILCOXONtest (one-sided) for the hypothesis of equal medians: Proportions of fetuses with malformations, variations and/or unclassified observations in each litter
Indices:
The calculations of the indices are given below in "Any other information on materials and methods".
- conception rate, preimplantation loss, postimplantation loss
Historical control data:
Historical control data are available for animals of this strain and age for parameters of conception rate, 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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
- occasional salivation: all high-dose group (1000 mg/kg bw/d) females and 3/25 of the middose group (300 mg/kg bw/d) females
- salivation occurred only shortly, i.e. within 0-2h, after treatment and was observed during GD 8-19 (test group 3) or only on GD 19 (test group 2)
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 clinical signs or changes of general behavior (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):
No test substance-related or spontaneous mortalities in any females of all test groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- mean body weights, average body weight gain of all dams (all dose groups) generally comparable to the concurrent control group throughout the entire study period
- indicental statistically significantly increased body weight gain value in test group 3 during GD 1-3
- corrected body weight gain of all test groups revealed no difference of any biological relevance; mean carcass weights of all test groups remained unaffected
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- mean food consumption of all test groups was generally comparable to the concurrent control group throughout the entire study period
- incidental statistically significantly increased food consumption value in test group 3 during GD 1-3
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
- mean gravid uterus weights of the animals of all test groups not influenced
- any differences between test groups and the control group revealed no dose-dependency and were assessed to be without biological
relevance
- mean placental weights of test groups 1-3 were comparable to the concurrent control group
Gross pathological findings:
no effects observed
Description (incidence and severity):
- no necropsy findings observed

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
- no test substance-related and/or biologically relevant differences between the different test groups for the pre- and post-implantation losses
Early or late resorptions:
no effects observed
Description (incidence and severity):
- no test substance-related and/or biologically relevant differences between the different test groups for the number of resorptions
Dead fetuses:
no effects observed
Description (incidence and severity):
- no test substance-related and/or biologically relevant differences between the different test groups for the number of viable fetuses
- two dead fetuses were found at cesarean section of one mid-dose dam (300 mg/kg bw/d); this rare finding may occur spontaneously in this rat strain
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
- conception rate was 92% in the control group and 100% in the low-, mid- and high-dose groups (100, 300 and 1000 mg/kg bw/d)
- no test substance-related and/or biologically relevant differences between the different test groups in conception rate,in the mean number of corpora lutea and implantation sites

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: no differences of toxicological relevance between the control and the treated groups for any reproductive parameters observed

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
- mean fetal weights of all test groups were not influenced by the test substance
- no biologically relevant differences in comparison to the control group
Changes in sex ratio:
no effects observed
Description (incidence and severity):
- sex distribution of the fetuses in all test group animals was comparable to the control fetuses
External malformations:
no effects observed
Description (incidence and severity):
- no external malformations recorded
- one external variation in one single fetus was recorded in the highest test group: limb hyperflexion; details given in table 1
- incidence not statistically significantly different from control and the finding was within the range of the historical control data; not considered as treatment-related and adverse
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- malformations noted in two fetuses, in test groups 1 and 3 (100 and 1000 mg/kg bw/d), individual details given in table 3 and total skeletal malformations given in table 4
- one female fetus of the high-dose group had multiple skeletal malformations concerning the skull, the vertebral column and ribs
- mean value of affected fetuses per litter was within the historical control range
- ‘fused mandible’ in one further individual fetus in the high-dose group; found in the historical control data in comparable incidences
- isolated findings not assessed as treatment-related and adverse
The total incidences of skeletal malformations in treated animals did not differ significantly from the control group and were comparable to the historical control data.

- skeletal variations of different bone structures were observed in all dose groups, with or without effects on corresponding cartilages, details for total fetal skeletal variations are given in table 5 and an overview of statistically significantly increased variations are given in table 6
- skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dose
- overall incidences of skeletal variations were comparable to the historical control data
- ‘incomplete ossification of skull’, ‘incomplete ossification of nasal’ and ‘supernumerary rib (14th); cartilage present’: not related to dose and the mean values were clearly inside the historical control ranges
- findings were not assessed as treatment related
- ‘supernumerary rib (14th); cartilage not present’: statistically significantly increased in test group 3 (affected fetuses/litter: 65.6%)
- value was marginally outside of the range of the historical control data
The rudimentary rib finding is very frequent in this rat strain and is not adverse per se. Since the finding was quite near the range of the historical control data, it was not assessed as treatment-related and adverse.
Visceral malformations:
no effects observed
Description (incidence and severity):
- no soft tissue malformations recorded
- four soft tissue variations were detected: short innominate in test groups 0 and 2, malpositioned subclavian origin in test group 1, dilated renal pelvis and dilated ureter in all test groups; details given in table 2
- dilated renal pelvis: mean values for affected fetuses per litter were outside the historical control range in the concurrent control and in the high-dose group alike
- values did, however, not significantly differ from each other; the finding was neither assessed as treatment-related nor as adverse
- dilated ureter: within the historical control range for all test groups
Overall, the incidences of all these variations were neither statistically significantly nor dosedependently increased in the treated groups.
Details on embryotoxic / teratogenic effects:
External and soft tissue malformations did not occur in any of the fetuses in this study. There were noted skeletal malformations in test groups 1 and 3 (100 and 1000 mg/kg bw/d).
One fetus of test group 3 carried multiple skeletal malformations (such as misshapen basisphenoid, cervical hemivertebra, thoracic hemivertebra with detached rib, misshapen thoracic vertebrae). Another fetus of test group 3 showed a fused mandible. Further malformations, i.e. absent lumbar vertebra and malpositioned and bipartite sternebra, were observed in individual fetuses, unrelated to dose.
All these findings were single cases, most of them can be found in the historical control data, except for ‘absent lumbar vertebra’. They also do neither form a pattern or syndrome with other minor anomalies which may raise toxicological concern nor do they influence the overall rate of malformations in this study.
There is no evidence for any association of these scattered findings with the treatment.
The total incidences of malformations are summarized in table 7.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose

Fetal abnormalities

Abnormalities:
effects observed, non-treatment-related

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Total external variations in one single fetus (no malformations) observed

 

 

Test group 0

0 mg/kgbw/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

23

232

25

250

25

228

25

255

 

Fetal incidence

 

N (%)

 

0.0

 

0.0

 

0.0

 

1 (0.4)

 

Litter incidence

 

N (%)

 

0.0

 

0.0

 

0.0

 

1 (4.0)

Affected fetuses/litter

 

Mean %

 

0.0

 

0.0

 

0.0

 

0.6

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

Table 2: Total soft tissue variations in the fetuses (no malformations) observed

 

 

Test group 0

0 mg/kgbw/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

23

111

25

119

25

108

25

121

Fetal incidence

 

N (%)

 

13 (12)

 

9 (7.6)

 

5 (4.6)

 

16 (13)

Litter incidence

 

N (%)

 

6 (26)

 

8 (32)

 

5 (20)

 

10 (40)

Affected fetuses/litter

 

Mean %

 

12.6

 

7.1

 

4.6

 

12.6

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

Table 3: Individual fetal skeletal malformations observed

Test group

Dam No.-Fetus No., Sex

Finding

0 (0 mg/kg bw/d)

none

 

1 (100 mg/kg bw/d)

28-08 M

absent lumbar vertebra

47-05 F

malpositioned and bipartite sternebra

2 (300 mg/kg bw/d)

none

 

3 (1000 mg/kg bw/d)

91-07 F

multiple skeletal malformations

97-09 M

fused mandible

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

Table 4: Total fetal skeletal malformations observed

 

 

Test group 0

0 mg/kgbw/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

23

121

25

131

25

120

25

134

Fetal incidence

 

N (%)

 

0.0

 

2 (1.5)

 

0.0

 

2 (1.5)

Litter incidence

 

N (%)

 

0.0

 

2 (8.0)

 

0.0

 

2 (8.0)

Affected fetuses/litter

 

Mean %

 

0.0

 

1.5

 

0.0

 

1.2

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

Table 5: Total fetal skeletal variations observed

 

 

Test group 0

0 mg/kgbw/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

23

121

25

131

25

120

25

134

Fetal incidence

 

N (%)

 

116 (96)

 

122 (93)

 

114 (95)

 

125 (93)

Litter incidence

 

N (%)

 

23 (100)

 

25 (100)

 

25 (100)

 

25 (100)

Affected fetuses/litter

 

Mean %

 

95.6

 

93.1

 

94.4

 

93.2

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

Table 6: Statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

 

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

 

4.1

 

10.7

 

12.6*

 

10.0

 

7.8

(2.1 - 15.9)

 

Incomplete ossification of nasal; unchanged cartilage

 

0.0

 

2.3*

 

0.0

 

0.8

 

0.8

(0.0 - 5.5)

 

Supernumerary rib (14th); cartilage present

 

2.4

 

3.5

 

8.7*

 

8.2*

 

7.8

(1.9 - 14.7)

 

Supernumerary rib (14th); cartilage not present

 

45.6

 

40.1

 

57.2

 

65.6*

 

54.0

(42.1 - 65.2)

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 7: Summary of the total fetal malformations observed

 

 

Test group 0

0 mg/kgbw/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

NN

23

232

25

250

25

228

25

255

Fetal incidence

 

N (%)

 

0.0

 

2 (0.8)

 

0.0

 

2 (0.8)

Litter incidence

 

N (%)

 

0.0

 

2 (8.0)

 

0.0

 

2 (8.0)

Affected fetuses/litter

 

Mean%

 

0.0

 

0.8

 

0.0

 

0.6

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

Applicant's summary and conclusion

Conclusions:
The oral administration of the test substance to 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/d 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/d.
Executive summary:

The test substance (UVCB) was tested for its prenatal developmental toxicity in Wistar rats according to OECD 414.

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 body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (drinking water; from day 8 onwards deionized water, with 5 mg/100 mL Cremophor EL) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, 23-25 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 in drinking water with 5 mg/100 mL Cremophor EL over a period of 7 days at room temperature was demonstrated and a homogeneous distribution of the test substance in the vehicle together with the correctness of the prepared concentrations was shown.

There were no test substance-related adverse effects of toxicological relevance on dams, gestational parameters or fetuses observed in any test group.

Based on these findings and under the conditions of the prenatal developmental toxicity study, the oral administration of the test substance to 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/d 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/d.