Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD 414 and GLP compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Physical state: Solid / light yellow
Expiry date: 25 Mar 2016
Storage conditions: Room temperature
Stability und er storage conditions: stable

Test animals

Species:
rat
Strain:
other: 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: About 10-12 weeks
- Weight at study initiation: 141.7 – 193.4 g
- Housing: single caging
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2013-02-13 To: 2013-03-12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% carboxymethylcellulose in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The specific amount of test substance was weighed, topped up with vehicle in a graduated flask and intensely mixed. The mixture was continuosly stirred until dosing.



VEHICLE
- Justification for use and choice of vehicle (if other than water): the test item can be suspended in the vehicle. It is not soluble in water.
- Amount of vehicle (if gavage): 10 ml/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HPLC-method
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
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.
Duration of treatment / exposure:
gestation days 6- 19
Frequency of treatment:
daily
Duration of test:
14 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100,300 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
25
Control animals:
yes
Details on study design:
- Dose selection rationale: Based on range-finder study with pregnant rats

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality/Morbidity, pertinent behavioral changes and/or signs of overt toxicity. were checked twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays (GO 0 to 20).

DETAILED CLINICAL OBSERVATIONS: No

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


POST-MORTEM EXAMINATIONS: Gross pathology
- Sacrifice on gestation day: GD 20

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: Site of implantations in the uterus
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [ half per litter ]
- Skeletal examinations: Yes: [ half per litter ]

In the present study the glossary of WISE et al. (1997) and its updated version of MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD et al. (1999) and SOLECKI et al. (2001, 2003):

Malformation
A permanent structural change that is likely to adversely affect the survival or health.

Variation
A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect the survival or health. This includes delays in growt or morphogenesis that have otherwise followed a normal pattern of development.

The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations.
Statistics:
DUNNETT's test: Food consumption, body weight, body weight change, DUNNETT's test
corrected body weight gain, carcass weight, weight of
the unopened uterus, weight of the placentas and
fetuses, corpora lutea, implantations, pre- and
postimplantation losses, resorptions and live fetuses

FISHER's exact test
Number of pregnant animals at the end of the study, FISHER's exact test mortality rate (of the dams) and number of litters with fetal findings

WILCOXON test
Proportion of fetuses with findings per litter
Indices:
sex ratio
conception rate (in %)
preimplantation loss (in %)
postimplantation loss (in %)
Historical control data:
Included in the final report.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
The mean food consumption of the dams in test groups 1-3 (100 or 300 mg/kg bw/d) was generally comparable to the concurrent control throughout the entire study period. The mean food consumption of the high-dose dams (1000 mg/kg bw/d) was slightly and temporarily, but statistically significantly reduced on GD 8-13. The accumulated food consumption of these dams during the treatment (GD 6-19) was comparable to the control. Thus, the temporary decrease on GD 8-13 may well have been an incidental event.
No further effects were recorded.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
See tables below.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
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

The stability of the test substance suspensions over a period of 7 days in a refrigerator was demonstrated. The homogeneous distribution of the test substance in the vehicle (1% CMC) was demonstrated. The results of the analysis of the aqueous test substance preparations confirmed the correctness of the prepared concentrations. The analytical values of the samples corresponded to the expected values within the limits of the analytical method, i.e. were always above 90% and below 110% of the nominal concentrations.

The mean gravid uterus weights of the animals of test group 1-3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance. No necropsy findings which could be attributed to the test substance were seen in any dam.

There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 100, 300 and 1000 mg/kg bw/d) in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation 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.

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. So were the mean placental weights and the mean fetal weights of the low-, mid- and high-dose groups. No fetus showed external malformations, variations or unclassified variations. Fetal data for soft tissue and skeletal examinations are summarized below.

Table 1: Individual fetal soft tissue malformation

Test group Dam No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) none  
1 (100 mg/kg bw/d) 27-02 M hydronephrosis, hydroureter
2 (300 mg/kg bw/d) none  
3 (1000 mg/kg bw/d) 86-08 F anophthalmia

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

Table 2: 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 N 25 25 25 25
Fetuses N 117 116 121 121
Fetal incidence N (%) 0.0 1 (0.9) 0.0 1 (0.8)
Litter incidence N (%) 0.0 1 (4.0) 0.0 1 (4.0)
Affected fetuses/litter Mean% 0.0 1.3 0.0 0.8

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

Table 3: Total fetal 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 N 25 25 25 25
Fetuses N 117 116 121 121
Fetal incidence N (%) 2 (1.7) 3 (2.6) 4 (3.3) 2 (1.7)
Litter incidence N (%) 2 (8.0) 3 (12) 4 (16) 1 (4.0)
Affected fetuses/litter Mean% 1.6 2.8 3.2 1.6

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

No unclassified soft tissue observations were recorded.

Table 4: Individual fetal skeletal malformations

Test group Dam No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) 6-01 M misshapen basioccipital
  12-05 F misshapen basioccipital
  12-09 F misshapen basisphenoid
  17-06 F misshapen basioccipital
1 (100 mg/kg bw/d) 43-01 M misshapen basisphenoid
  47-06 F small cervical arch
2 (300 mg/kg bw/d) 58-07 M fused thoracic centrum, fused rib
  71-10 F severely malformed vertebral column
3 (1000 mg/kg bw/d) 82-04 M shortened humerus
  86-06 F exoccipital fused with 1stcervical arch, misshapen basioccipital, cervical hemivertebra
  89-09 F absent lumbar vertebra, intercostal rib, branched rib

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

Table 5: Total fetal 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 N 25 25 25 25
Fetuses N 132 128 133 130
Fetal incidence N (%) 4 (3.0) 2 (1.6) 2 (1.5) 3 (2.3)
Litter incidence N (%) 3 (12) 2 (8.0) 2 (8.0) 3 (12)
Affected fetuses/litter Mean% 3.0 1.5 1.6 2.5

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

Table 6: 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 N 25 25 25 25
Fetuses N 132 128 133 130
Fetal incidence N (%) 127 (96) 127 (99) 132 (99) 129 (99)
Litter incidence N (%) 25 (100) 25 (100) 25 (100) 25 (100)
Affected fetuses/litter Mean% 96.5 99.0 99.2 99.3

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

Table 7: Occurrence of 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 cervical arch; cartilage pre- sent 0.0 0.8 2.1* 3.0* 0.1
(0.0 – 0.8)
Incomplete ossification of thoracic centrum; unchanged cartilage 0.0 1.8 2.4* 1.5 1.0
(0.0 – 4.8)
Incomplete ossification of sacral arch;
cartilage present
1.2 3.7 2.8 7.3* 1.5
(0.0 – 6.0)
Unossified sternebra; unchanged cartilage 4.0 10.7* 5.3* 8.7 8.6
(2.6 – 20.7)
Unilateral ossification of sternebra;
unchanged cartilage
1.0 2.0 0.8 4.9* 1.2
(0.0 – 4.0)

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

* = p ≤ 0.05 (Wilcoxon-test [one-sided])

All these minor changes may be indicative for a minimal delay of skeletal maturation, however, the slightness of these apparent effects makes it difficult to judge whether these are real effects or not. They are in any case not considered as adverse events.

Table 8: 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 N 25 25 25 25
Fetuses N 132 128 133 130
Fetal incidence N (%) 98 (74) 88 (69) 88 (66) 99 (76)
Litter incidence N (%) 25 (100) 25 (100) 25 (100) 25 (100)
Affected fetuses/litter Mean% 73.9 68.7 66.9 75.9

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

Applicant's summary and conclusion

Conclusions:
The substance does not cause developmental toxicity or teratogenicity in rats.
Executive summary:

In a prenatal developmental toxicity study the test substance was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental

toxicity. Analyses confirmed the correctness of the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle.

Generally, clinical observations including food consumption and body weight gain revealed no toxicologically relevant difference between the animals receiving 100, 300 or 1000 mg/kg bw/d test substance and controls. No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/d) were determined for any reproductive parameters, such as

conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.