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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:
2001
Report date:
2001

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(1981)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: 20% of the pure active substance in water
Details on test material:
- Name of test material: DOPA-Glycinate

Test animals

Species:
rat
Strain:
other: Wistar rats Crl: (WI) BR (outbred, SPF-Quality)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: Approx. 10 weeks
- Weight at study initiation: 221–310 g
- Housing: in groups of 5 during acclimatisation, durung mating females were caged together with males on a one-to-one-basis; individually during study
- Diet (e.g. ad libitum): standard pelleted laboratory animal diet (from Car-51 Quality BVBA, Oud-Turnhout, Belgium), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 12 d prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-100
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the formulations revealed values for accuracy within the range of 99% and 103% of nominal for the week 1 formulations, and between 88% and 119% of nominal for the week 3 formulations. This was considered to represent an acceptable level for formulations of this type.
The low and high dose formulations were considered to be stable over 4 hours and sufficiently homogenous.
Method of analysis: TOC analysis; this is considered suitable in view of the fact that the test substance was the only organic constituent in the dosing solutions.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Once mating had occurred, the female was separated from the male and vaginal smearing was discontinued. When sufficient mated females were obtained for each dose group, the surplus females were removed from the study.

Duration of treatment / exposure:
Day 6–16 of gestation
Frequency of treatment:
daily
Duration of test:
21 d
Doses / concentrationsopen allclose all
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Remarks:
based on test material (20% formulation)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
based on test material (20% formulation)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
based on test material (20% formulation)
Dose / conc.:
8 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels for the present embryotoxicity and teratogencity study were based on a preliminary study in pregnant rats.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- mortality/viability: twice daily

BODY WEIGHT: Yes
On day 0 and 3, daily from day 6 to day 17 inclusive and on day 21 post-coitum

FOOD CONSUMPTION: Yes
during days 0–3, 3–6, 6–9, 9–13, 13–17 and 17–21 post-coitum

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21
ovaries and uterine content
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
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups.
The Steel-test (many-to-one rank test) was applied instead of the Dunnett-test if the data can not be assumed to follow a normal distribution.
The exact Fisher-test was applied for 2x2 tables if variables could be dichotimized without loss of information.
The Student’s t test was applied on placenta data to test the difference between the treatment and the control groups.
All tests will be two-sided and in all cases p < 0.05 were accepted as the lowest level of significance.
Indices:
Implantation index, Implantation site scar index, Embryonic/foetal death index, Embryonic resorption index, Foetal resorption index, Total foetus index, Live foetus index, Dead foetus index, Abnormal foetus index, Percentage males, Percentage females, Percentage live males, Percentage live females

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Four animals treated with 250 mg/kg bw/day showed incidental findings (hunched posture, laboured respiration, rales, brown discolouration of the snout, salivation and piloerection) which were however considered to be related to treatment. One animal treated with 40 mg/kg bw/day showed scabs on the nose and one which received 100 mg/kg bw/day showed piloerection. These effects were considered not to be treatment related. Alopecia was observed in all animals of all dose groups. Alopecia is commonly seen in animals of this strain used in this type of study and therefore no toxicological significance was attached to this finding.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females receiving 250 mg/kg bw/day showed a decrease in body weight and body weight gain, which was statistically significant on gestation days 13 to 17 and 11 to 17, when compared to the control group. After correction of the uterus weight, a decrease in body weight gain was noted in the 250 mg/kg bw/day dose group when compared to the control. However, this was not statistically significant due to the high standard deviation in this treated group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females treated with 250 mg/kg bw/day showed a statistically significant decrease in food consumption on gestation days 6 to 17. This finding was considered to be caused by the test substance. Females receiving 100 mg/kg bw/day showed a slight, statistically significant decrease in food consumption from gestation days 6–9 and 13–17 when compared to the control. However, this difference was considered to be unrelated to treatment because the mean of relative food consumption of females of this dose group were comparable with the control values.
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):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related macroscopic lesions were observed upon necropsy.
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:
effects observed, non-treatment-related
Description (incidence and severity):
The statistically significant differences seen in pre-implantation loss and implantation index of the 40 and 250 mg/kg bw/d dose group were considered to be unrelated to treatment, since treatment started on gestation day 6, which is after implantation. No treatment related effects were seen on post-implantation loss.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
One dead foetus was noted in the 40 mg/kg bw/day dose group upon caesarean sectioning. The number of live foetuses observed in the treatment and control groups remained within biologically normal limits for rats of this age and strain and no treatment related differences were observed.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
With the exception of one female of the 100 mg/kg bw/day dose group, all females were pregnant. The non-pregnant female was excluded from further calculations.
Other effects:
not examined
Details on maternal toxic effects:
Results are presented in Table A6.8.1- 5 and Table A6.8.1- 6.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Foetal weights and of dams and placental weights of live foetuses of the 40 mg/kg bw/d group were significantly decreased when compared to the control group. Due to absence of a dose-relation, this change was considered to be unrelated to treatment.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
The number of live foetuses observed in the treatment and control groups remained within biologically normal limits for rats of this age and strain and no treatment related differences were observed.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
External examination of the foetuses did not reveal any findings among foetuses of litters treated with the test substance that were considered to be an adverse effect of the test substance. There are minor morphological changes amongst all litters across all dose groups as well as the control. In general there were no indications of any consistent treatment or dose related effects on any of the parameters investigated, some of which are tabulated in A6.8.1-4 for illustrative purposes. Malrotated limbs, and small foetuses were noted in all groups. Incidental findings consisted of two very
autolysed foetuses (of which one foetus was dead), and an umbilical hernia
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Compared with the concurrent controls and the 40 mg/kg bw/day dose group, there appeared to be indications of a very marginal reduction in ossification in the 250 mg/kg bw/day dose group. Parameters affected included a number of cranial bones, viz. supraoccipital, interparietal, squamosal, jugal, maxilla(e) and hyoid, the caudal vertebral arches and humerus(i). Other ossification parameters were similar in all groups.
However, reference to laboratory historical control data indicated that the concurrent control values showed a slightly advanced ossification, whilst the majority of the values in the 100 and 250 mgkg bw/day dose groups were within the historical control ranges.
It was considered, therefore, that the slight differences from the concurrent controls were of no toxicological significance.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A number of anomalies were recorded in all groups, the types, incidences and group distribution of which did not indicate any association with treatment.
In the 100 and 250 mg/kg bw/day dose groups there appeared to be a marginal increase in the incidence of unilateral hydroureter compared with the concurrent controls and the 40 mg/kg bw/day dose group, however, as no increases were recorded in the incidences of bilateral hydroureter or renal pelvic cavitation it was considered that this finding did not represent a toxicologically significant response to treatment.
In the 250 mg/kg bw/day dose group one litter contained four foetuses with an accessory blood vessel associated with the superior pole of the kidney and a second litter contained one small foetus with cranial abnormalities and immature gonads. The isolated nature of these findings did not suggest any association with treatment.
Other effects:
not examined

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
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 A6.8.1-3: Rat Maternal and Foetal Observations made at necropsy.

Parameter

Control

40 mg/kg/d

100 mg/kg/d

250 mg/kg/d

Number of dams examined

24

24

23a

24

Mortality of dams

0

0

0

0

Mean body weight [g] at day 0

262

267

256

261

Mean body weight [g] at day 17

364

377

352

341**

Mean body weight [g] at day 21

439

455

423

418

Mean body weight gain [%] day12

23

24

23

19**

Mean body weight gain [%] day 17

39

41

38

31**

Mean food consumption [g/rat/day], day 6–9

27

28

25*

23**

Mean food consumption [g/rat/day], day 9–13

28

28

26

22**

Mean food consumption [g/rat/day], day 13–17

29

30

26*

23**

Relative food consumption [g/kg bw/day], day 13–17

85

86

80

70**

Number of pregnant females

24/24

24/24

23/24

24/24

Abortions

0

0

0

0

 

 

 

 

 

No. of litters totally resorbed

0

0

0

0

No. of litters with viable foetuses

24

24

23

24

No. of corpora lutea/dam

18.6 ± 2.8

19.7 ± 2.9

17.9 ± 2.8

17.8 ± 2.1

No. of implantations/dam

15.5 ± 2.9

17.3 ± 2.4

15.0 ± 2.8

16.0 ± 2.6

Mean% pre-implantation loss

16.6

11.9#

16.5

10.1##

No. of resorptions/litter

0.5

0.5

0.6

0.9

Mean% post-implantation loss

3.5

2.9

4.1

5.5

Viable foetuses (total)

359

404

330

363

Viable foetuses/litter

15.0 ± 2.9

16.8 ± 2.3

14.3 ± 3.0

15.1 ± 3.2

Dead foetuses (total)

0

1

0

0

Foetal weight-males (g)

5.3 ± 0.5

5.3 ± 0.6

5.4 ± 0.4

5.4 ± 0.5

Foetal weight-females (g)

5.0 ± 0.5

4.9 ± 0.8

5.1 ± 0.4

5.1 ± 0.5

Foetal weight-sexes combined (g)

5.2 ± 0.5

5.1 ± 0.7

5.2 ± 0.4

5.2 ± 0.5

Sex ratio (M%:F%)

48:52

48:52

50:50

52:48

Mean placental weight (g)

0.51 ± 0.06

0.47 ± 0.06

0.53 ± 0.06

0.50 ± 0.06

 

 

 

 

 

a) one animal (female 51) did not become pregnant after mating.

* : Dunnett-test based on pooled variance significant at 5% level

** : Dunnett-test based on pooled variance significant at 1% level

#: Fisher’s Exact Test significant at level 5%

##: Fisher’s Exact Test significant at level 1%

 

Table A6.8.1-4: Incidence of Selected Rat Foetal Parameters*(Caesarean section data from day 21).

Parameter

Control

40 mg/kg/d

100 mg/kg/d

250 mg/kg/d

 

 

Number of foetuses (no. of litters) examined

visceral examination

182 (24)

201 (24)

162 (23)

180 (24)

 

skeletal examination

185 (24)

202 (24)

168 (23)

183 (24)

 

Visceral Examination - affected foetuses (no. of litters)

 

small additional vessel arising from aorta

1 (1)

0 (0)

0 (0)

0

 

small additional liver lobe

14 (12)

12 (9)

14 (7)

14 (11)

 

kidney and adrenal gland displaced

7 (6)

9(5)

4(4)

5(5)

 

↑ renal pelvic cavitation: unilateral

0 (0)

0 (0)

1 (1)

2 (2)

 

↑ renal pelvic cavitation: bilateral

1(1)

0 (0)

0 (0)

0 (0)

 

hydroureter: unilateral
           % of total foetuses:

4 (3)
2.2

5 (4)
2.5

12 (9)
7.1

17 (9)
9.3

 

hydroureter: bilateral

4 (2)

4 (3)

0 (0)

6 (5)

 

Skeletal Examination - affected foetuses (no. of litters)

 

delayed ossification interparietal
           % of total foetuses:

23 (13)
12.4

23 (13)
11.4

31 (10)
18.5

42 (13)
23.0

 

delayed ossification supraoccipital
           % of total foetuses:

4 (2)
2.2

7 (5)
3.5

12 (7)
7.1

19 (10)
10.4

 

delayed ossification hyoid

2 (2)

1 (1)

8 (6)

6 (3)

 

delayed ossification sternebrae (≥3)

2 (2)

3 (3)

1 (1)

6 (5)

 

anomalous sternabrae

9 (6)

7 (7)

7 (7)

14 (11)

 

Head Examination – affected foetuses (no. of litters)

 

bilateral slightly folded retina

1 (1)

0 (0)

0 (0)

1 (1)

 

 

 

 

 

 

 

*Some of the effects seen multiple times at any dose are recorded here. There was no consistent relationship between dose and any of the recorded effects noted in the original pathologist‘s report except for the incidence of hydroureter. Numbers in parenthesis indicate the number of litters in which that effect is found. 

 

 

Visceral Examination

A number of anomalies were recorded in all groups, the types, incidences and group distribution of which did not indicate any association with treatment.

In the intermediate and high dose groups (100 and 250 mg/kg body weight/day) there appeared to be a marginal increase in the incidence of unilateral hydroureter compared with the concurrent controls and the low dose group (40 mg/kg body weight/day), however, as no increases were recorded in the incidences of bilateral hydroureter or renal pelvic cavitation it was considered that this finding did not represent a toxicologically significant response to treatment.

In the high dose group (250 mg/kg body weight/day) one litter contained four fetuses with an accessory blood vessel associated with the superior pole of the kidney and a second litter contained one small foetus with cranial abnormalities and immature gonads. The isolated nature of these observations did not suggest any association with treatment.

 

Skeletal Examination

Compared with the concurrent controls and the low dose group (40 mg/kg body weight/day), there appeared to be indications of reduction in foetal ossification in the high dose group (250 mg/kg body weight/day) and, to a lesser extent, in the intermediate dose group (100 mg/kg body weight/day). Parameters affected included a number of cranial bones, viz. supraoccipital, interparietal, squamosal, jugal. maxilla(e) and hyoid, the caudal vertebral arches and humerus(i). Other ossification parameters were similar in all groups. However, reference to laboratory historical control data indicated that the concurrent control values showed a slightly advanced ossification, whilst the majority of the values in the 100 and 250 mgkg dose groups were within the historical control ranges. It was considered, therefore, that the slight differences from the concurrent controls were of no toxicological significance.

 

Morphological parameters

A number of foetuses in all groups showed minor morphological changes, but there were no indications of any consistent treatment or dose related associations or trends.

Applicant's summary and conclusion

Conclusions:
Dams treated with 250 mg/kg bw/day registration substance (20% a.i.) showed maternal toxicity comprising a decrease in body weight and body weight gain, which were statistically significant on gestation days 13 to 17 and 11 to 17, when compared to the control group. Females treated with 250 mg/kg bw/day showed a statistically significant decrease in food consumption on gestation days 6 to 17. Four animals out of 24 in the high dose group showed generalised clinical findings including hunched posture, laboured respiration, rales, brown discolouration of the snout, salivation and piloerection. The maternal NO(A)EL is set at 100 mg/kg bw/day (corresponding to 20 mg a.i. /kg bw/day).

External, visceral, skeletal examination of the foetuses did not reveal any findings among foetuses of litters treated with the test substance that were considered to be an adverse effect of the test substance. Oral administration of the registration substance (20% a.i.) to pregnant Wistar rats during the period of organogenesis at dose levels up to 250 mg/kg bw/day (corresponding to 50 mg a.i./kg bw/day) did not result in any adverse effects during foetal development.
Executive summary:

The potential of the registration substance (20% a.i.) to induce embryotoxic effects in the rat (Wistar rats Crl: (WI) BR (outbred, SPF-Quality)) was investigated at 0 (control), 40, 100 and 250 mg/kg bw/day in water, administered from day 6 to day 16 post coitum, according to OECD guideline 414 (1981). Furthermore, the conduct of the study was consistent to the recent OECD guideline 414 (2001) in all important aspects, except that test substance was administered solely during the period of organogenesis.

 

Visceral Examination

A number of anomalies were recorded in all groups, the types, incidences and group distribution of which did not indicate any association with treatment.

In the intermediate and high dose groups (100 and 250 mg/kg body weight/day) there appeared to be an increase in the incidence of unilateral hydroureter compared with the concurrent controls and the low dose group (40 mg/kg body weight/day), however, as no increases were recorded in the incidences of bilateral hydroureter or renal pelvic cavitation it was considered that this finding did not represent a toxicologically significant response to treatment.

In the high dose group (250 mg/kg body weight/day) one litter contained four fetuses with an accessory blood vessel associated with the superior pole of the kidney and a second litter contained one small foetus with cranial abnormalities and immature gonads. The isolated nature of these observations did not suggest any association with treatment.

 

Skeletal Examination

Compared with the concurrent controls and the low dose group (40 mg/kg body weight/day), there appeared to be indications of a reduction in foetal ossification in the high dose group (250 mg/kg body weight/day) and, to a lesser extent, in the intermediate dose group (100 mg/kg body weight/day). Parameters affected included a number of cranial bones, viz. supraoccipital, interparietal, squamosal, jugal. maxilla(e) and hyoid, the caudal vertebral arches and humerus(i). Other ossification parameters were similar in all groups.

 

Morphological Examination

A number of foetuses in all groups showed minor morphological changes, but there were no indications of any consistent treatment or dose related associations or trends.

 

Dams treated with 250 mg/kg bw/d showed maternal toxicity comprising clinical signs, reduced body weight gain and food consumption during the treatment period. Based on the results of this study, DOPA-Glycinate (20% a.i.) is considered not to be teratogenic when orally administered to Wistar rats at levels up to and including 40 mg/kg bw/d.

The maternal no observed adverse effect level (NOAEL) was 100 mg/kg bw/d, corresponding to 20 mg a.i./kg bw/d.

The NOAEL for survival, growth and development in utero was 40 mg/kg bw/d, corresponding to 8 mg a.i./kg bw/d.