Registration Dossier

Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Prenatal developmental toxicity studies on diundecyl and ditridecyl phthalates in Sprague-Dawley rats
Author:
Saillenfait A-M
Year:
2013
Bibliographic source:
Reproductive Toxicology 37 (2013) 49–55

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
not specified
Remarks:
(this information was not provided)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Diundecyl phthalate
EC Number:
222-884-9
EC Name:
Diundecyl phthalate
Cas Number:
3648-20-2
Molecular formula:
C30H50O4
IUPAC Name:
1,2-diundecyl benzene-1,2-dicarboxylate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Saint-Germain-sur-l’Arbresle, France)
- Weight at study initiation: ca. 230 g average
- Housing: Mated females were singly housed in clear polycarbonate cages with stainless steel wire lids and virgin loose pulp as bedding (Alpha-Dri®, Dietex, Saint Gratien, France).
- Diet (e.g. ad libitum): Food pellets (UAR Alimentation, Villemoisson, France), ad libitum.
- Water (e.g. ad libitum): filtered tap water, ad libitum.
- Acclimation period: 1-2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 15 %
- Photoperiod (hrs dark / hrs light): 12 light / 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations were prepared weekly and stored in a dark place at room temperature. Stability was established for up to 2 weeks by gas chromatography analysis.

VEHICLE
- Concentration in vehicle: 0, 50, 100, 200 mg/mL, giving dose levels of 0, 250, 500, 1000 mg/kg/day.
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability was established for up to 2 weeks by gas chromatography analysis.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: primiparous female (180–200 g) rats were housed overnight with adult males from the same strain and supplier.
- M/F ratio per cage: not specified
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of gestation.

Duration of treatment / exposure:
Days 6 - 20 of gestation
Frequency of treatment:
Daily
Duration of test:
From day 0 to day 21 of gestation, when dams were euthanized.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
21-22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dosage levels of the definitive study were based on the findings of a dose-range finding study in which pregnant rats (9 or 10/group) were given 0, 0.5 or 1 g/kg/day of DUDP by gavage, on GD 6 to GD 20.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Maternal body weights were recorded on GD 0, 6, 9, 12, 15, 18, and 21.

FOOD CONSUMPTION: Yes, food consumption recordings were made at 3 day intervals commencing on GD 6.

POST-MORTEM EXAMINATIONS: No data other than for uterine contents.

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 and late resorptions: Yes
- Number of distribution of foetuses in each uterine horn: yes
Fetal examinations:
- External examinations: Yes - all per litter
- Soft tissue examinations: Yes - half per litter
- Skeletal examinations: Yes - half per litter
- Head examinations: Yes - half per litter (as part of soft tissue examination)
- Other: Anogenital distance (AGD) was measured using a dissecting microscope with a micrometer eyepiece. The degree of trans-abdominal testicular migration (TTM) was determined by measuring the distance from the bladder neck to the lower pole of the testes using a dissecting microscope with a micrometer eyepiece.

Statistics:
The litter was used as the basis for the analysis of foetal variables.

Maternal body weights, body weight gain and food consumption of pregnant rats, number of corpora lutea, number of implantation sites and live foetuses, litter mean foetal body weight and AGD were analysed by one-way analysis of variance, followed by Dunnett’s test if differences were found.

The mean percentage of post-implantation loss, dead foetuses, resorptions, sex ratio (male foetuses per litter), and the proportion of affected foetuses per litter (calculated for each alteration) were evaluated by using the Kruskal–Wallis test, followed by the Mann–Whitney test if differences were indicated.

The rate of pregnancy and the number of litters with dead foetuses, resorptions, or foetal alterations were analysed by using Fisher’s test. Additional statistical evaluations were performed using the number of live foetuses per litter as a covariate for foetal body weight. Foetal AGD was also analysed with foetal body weight as covariant, using a linear mixed-effects model with two levels of variance in which litter effect was modelled with a nested random factor and dose and foetal weights as fixed factors. Least-squares analysis was carried out where applicable.

Treated groups were compared to their respective vehicle control.

All tests were reported at the 5% or 1% level of significance.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were noted.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All pregnant animals survived to scheduled euthanization on GD 21.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant changes in mean maternal body weights and body weight gains throughout the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Maternal food consumption was unaffected by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
Treatment did not influence drinking-water consumption.
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:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were registered either in treated groups or in the control group.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
No significant differences were observed in the number of corpora lutea or incidence of pre-implantation loss. The number of implants was slightly but significantly lower than the concurrent control at the low and mid doses. There were no effects of DUDP on post-implantation loss.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no effects of DUDP on resorptions.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no effects of DUDP on resorptions.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no effects of DUDP on live fetuses.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No further influence on the prenatal development was detected.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
LOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: decrease in implantation sites
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal general toxicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no effects of DUDP on fetal body weights.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no effects of DUDP on live fetuses.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no effects of DUDP on fetal sex ratio (percent male fetuses per litter).
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations occurred in one fetus at 0.25 g/kg/day (omphalocele) and in one fetus at 1 g/kg/day (diaphragmatic hernia). These isolated cases were considered incidental. Common external (i.e. club foot) variations were seen in single or few fetuses, with no indication of any adverse effects related to the administration of DUDP.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
An increased occurrence of lumbar ribs was observed in fetuses from the 0.5 and 1 g/kg/day dose groups, compared to control. The mean percentage of affected fetuses per litter amounted to 10.3, 20.8, 46.6, and 25.4 at 0, 0.25, 0.5, and 1 g/kg/day, respectively. The historical control range was from 6.8% to 19.4%. The incidence of affected litters was also significantly increased at the mid dose.

Long supernumerary ribs were only observed in one fetus at 0.25 g/kg/day and in one fetus at 1 g/kg/day. Otherwise, supernumerary 14th ribs were pinpoint ossification sites (78–88%) or were short, in the control and treated groups. There were no significant changes in the incidences of any other skeletal variations or in the ossification of metacarpals, metatarsals, and phalanges. The elevated number of ossified caudal vertebral centra in the DUDP-treated groups compared to control was not considered toxicologically meaningful. All fetuses had 26 presacral vertebrae.

Although the increase in the incidence of fetuses with short supernumerary lumbar ribs at 0.5 and 1 g DUDP/kg/day did not occur in a clear dose-related manner a relationship to treatment cannot be ruled out, and also the authors state that supernumerary lumbar ribs, especially longer ribs, could be regarded as being an indicator of changes in axial skeletal development. However, rudimentary supernumerary ribs are usually considered as common reversible variants in rodent bioassays and there were no other notable skeletal findings at any dose investigated. Thus, it is considered that no adverse effects were found up to the highest dose tested.

Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Common visceral (e.g. left umbilical artery) variations were seen in single or few fetuses, with no indication of any adverse effects related to the administration of DUDP.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Male AGD was identical to control at 0.25 g/kg/day, and it was slightly, although not significantly, reduced at 0.5 and 1 g/kg/day (3–4%). A statistically significant difference was only noted at 0.5 g/kg, after adjustment with the cubic root of fetal weight or when fetal body weight was used as covariate (with litter based analysis or mixed-effects model).

There was no male with mal-positioned testis and the pattern of trans-abdominal testicular migration was comparable across groups.

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
LOEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect observed at the highest dose tested

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. Maternal findings.

 

DUDP (g/kg/day)

 

0

0.25

0.50

1.00

No. dead/treated 

0/22

0/21

0/21

0/22

No. (%) pregnant

22 (100.0)

21(100.0)

21(100.0)

20(90.9)

Body weight (g)

 

 

 

 

GD 0 

230±13a

229±11

229±13

231±13

GD 6 

259±11

259±11

258±15

257±15

GD 9 

272±11

273±12

274±18

269±15

GD 12 

290±12

290±14

291±21

286±17

GD 15 

312±15

311±14

314±24

309±18

GD 18 

354±19

352±16

353±33

352±20

GD 21 

414±25

407±25

405±47

408±27

Gravid uterine weight (g)

107±13

98±23

96±29

103±15

Net body weight (g)b

307±16

309±17

309±25

305±20

Body weight change (g)

 

 

 

 

GD 0–6 

29±6

30±6

29±8

26±6

GD 6–9 

14±3

14±5

16±4

11±5

GD 9–12 

18±5

17±5

17±8

17±4

GD 12–15 

22±5

21±6

23±9

24±4

GD 15–18 

42±6

42±8

39±13

43±7

GD 18–21 

60±9

55±11

52±16

56±11

GD 0–21 

184±19

178±22

177±43

177±21

Net weight gain (g)c

78±14

80±12

81±23

74±16

Food consumption (g/day)

 

 

 

 

GD 0–6 

21±1

21±2

21±2

21±2

GD 6–9 

19±2

21±2

21±3

21±4

GD 9–12 

21±2

23±2

23±3

22±3

GD 12–15 

22±2

23±2

24±4

23±2

GD 15–18 

24±2

26±2

26±4

27±2

GD 18–21 

25±3

25±3

25±5

26±3

GD 0–21 

22±1

23±2

23±3

23±2

Body weight on GD 21 (b) or body weight gain during GD 0–21 (c) minus gravid uterine weight.

a Values are expressed as means±SD.

* Significant differences from the vehicle control, p < 0.05.

** Significant differences from the vehicle control, p < 0.01.

Table 2. Reproductive findings.

 

DUDP (g/kg/day)

 

0

0.25

0.50

1.00

All litters a  

22

21

21

20

No. corpora lutea 

15.7±1.5b

14.7±2.0

14.9±1.8

15.3±1.7

%Pre-implantation loss per litter

3.0±4.8

10.4±17.7

8.4±13.7

5.9±10.2

No. implantation sites per litter

15.2±1.8

13.2±3.3*

13.4±2.9*

14.3±1.7

%Post-implantation loss per litter c

6.4±10.8

2.5±5.9

8.0±21.4

5.6±5.7

No. litters with dead fetus

1

1

1

0

%Dead fetuses per litter

0.3±1.2

0.4±1.8

0.3±1.5

0.0±0.0

No. litters with resorptions

12

5

9

6

%Resorptions per litter 

6.1±10.7

2.2±4.4

7.7±21.5

3.7±7.2

Live littersd  

22

21

20

20

No. live fetuses per litter

14.2±1.9

12.9±3.3

13.2±2.7

13.8±2.0

%Male fetuses per litter

48.4±12.1

51.1±19.5

42.9±13.7

49.9±15.8

Fetal body weight (g)

 

 

 

 

All fetuses  

5.52±0.31

5.58±0.36

5.60±0.21

5.53±0.27

Male fetuses  

5.69±0.31

5.75±0.34

5.78±0.25

5.69±0.30

Female fetuses  

5.37±0.32

5.37±0.39

5.48±0.19

5.39±0.28

AGD (mm)  

 

 

 

 

No. litters  

18

17

16

16

Male fetuses  

2.96±0.12

2.95±0.15

2.85±0.11

2.86±0.15

Female fetuses  

1.04±0.06

1.06±0.06

1.07±0.06

1.09±0.06

AGD/body weight 1/3  

 

 

 

 

Male fetuses  

1.65±0.08

1.65±0.08

1.59±0.05*

1.60±0.09

Female fetuses  

0.59±0.03

0.60±0.03

0.61±0.03

0.62±0.04

a Includes all pregnant females at euthanization.

b Values are expressed as means±SD.

c [(No. resorptions plus dead fetuses)/No. Implantations]×100.

d Includes all animals with live fetuses at euthanization.

* Denotes significant differences from the vehicle control, p < 0.05.

Table 3. Fetal malformations and variations

 

Dose (g/kg/day)

 

0

0.25

0.50

1.00

No. fetuses (litters) examined a

 

 

 

 

External

312 (22)

270 (21)

264 (20)

275 (20)

Visceral

156 (22)

135 (21)

132 (20)

138 (20)

Skeletal

156 (22)

135 (21)

132 (20)

137 (20)

Malformations

 

 

 

 

Omphalocele

0

1 (1)

0

0

Diaphragmatic hernia

0

0

0

1 (1)

External variations

 

 

 

 

Club foot (unilateral)

0

0

1 (1)

0

Visceral variations

 

 

 

 

Umbilical artery, left

4 (4)

2 (2)

4 (4)

4 (3)

Distended ureter

1 (1)

0 (0)

0 (0)

0 (0)

Skeletal variations

 

 

 

 

Sternebra ossification

 

 

 

 

Dumbell, bipartite, incomplete or absent (5th)

2 (2)

3 (2)

2 (2)

0

Misaligned

1 (1)

0

0

0

Cervical rib(s)

2 (1)

3 (3)

2 (1)

4 (3)

14th rib(s), supernumerary (any)

17 (10)

29 (13)

60 ##(17 )*

32 # (12)

Long b

0

0

1 (1)

1 (1)

Thoracic vertebral centra, ossification

 

 

 

 

Bipartite, dumbbell and/or incomplete (one or two)

13 (8)

16 (11)

20 (10)

16 (7)

Lumbar vertebral centra, ossification, bipartite or incomplete

0

0

1 (1)

1 (1)

No. ossification centres

 

 

 

 

Metacarpals

4.00 ± 0.00 c

3.99 ± 0.05

4.00 ± 0.00

4.00 ± 0.00

Forelimb proximal phalanges

3.79 ± 0.33

3.67 ± 0.67

3.74 ± 0.29

3.81 ± 0.36

Metatarsals

4.95 ± 0.08

4.96 ± 0.16

4.99 ± 0.04

5.00 ± 0.01

Hindlimb proximal phalanges

2.26 ± 0.95

2.33 ± 1.12

2.15 ± 0.92

2.38 ± 1.19

Caudal vertebral centra

6.12 ± 0.37

6.59 ± 0.70 §

6.70 ± 0.57 §§

6.67 ± 0.65 §§

a The incidence of individual defect is presented as number of fetuses (number of litters).

b More than one third of the length of the preceeding rib

c Mean±SD.

* Denotes significant differences from control, p < 0.05 (Fisher’s test).

# Denotes significant differences from control, p < 0.05, (Mann–Whitney test).

## Denotes significant differences from control, p < 0.01, (Mann–Whitney test).

§ Denotes significant differences from control, p < 0.05, (Dunnett’s test).

§§ Denotes significant differences from control, p < 0.01 (Dunnett’s test).

Applicant's summary and conclusion

Conclusions:
In a pre-natal developmental toxicity on diundecyl phthalate (DUP) no evidence of teratogenic effects was observed after oral administration at levels up to 1000 mg/kg bw/day.

Executive summary:

A pre-natal developmental toxicity test was performed with DUP following a method similar to OECD Guideline 414. Groups of 21-22 pregnant Sprague-Dawley rats were administered 0, 250, 500, or 1000 mg/kg/day of DUDP diluted in olive oil by gavage on gestation days 6–20. DUDP had no adverse effects on maternal body weight and food consumption. The number of live fetuses, percent of post-implantation loss and resorptions, fetal sex, and fetal body weights were not affected. A statistically significant decrease in implantation sites was observed at doses of 250 and 500 mg/kg/day. The significance of this observation was considered unclear and authors suggest that female fertility and reproduction need to be further investigated. Small decreases in the anogenital distance of male fetuses were noted at 500 and 1000 mg/kg/day. This finding provides limited evidence that DUDP might have marked effects on the male sexual differentiation and thus further studies are needed to be conducted to evaluate the effects of an in utero exposure on the male sexual development in the fetal, young, and adult offspring. No evidence of teratogenic effects was observed after oral administration at levels up to 1000 mg/kg bw/day. There were no significant changes in the incidence of external, visceral or skeletal variations, except for an increase in the incidence of foetuses with short supernumerary lumbar ribs at 500 and 1000 mg/kg bw/day. Although this variation did not occur in a clear dose-related manner, a relationship to treatment cannot be ruled out and the authors state that supernumerary lumbar ribs, especially longer ribs, could be regarded as being an indicator of changes in axial skeletal development. However, rudimentary supernumerary ribs are usually considered as common reversible variants in rodent bioassays and there were no other notable skeletal findings at any dose investigated. The NOAEL is therefore regarded as being 1000 mg/kg bw/day, the highest dose investigated.