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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Peer reviewed literature report of a study conducted to recognised international test guidelines. For read-across justification see Section 13.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diundecyl phthalate (DUDP)
- Analytical purity: > 98%

- Molecular formula (if other than submission substance): C30 H50 O4
- Molecular weight (if other than submission substance): 474.73
- Smiles notation (if other than submission substance): O=C(OCCCCCCCCCCC)c(c(ccc1)C(=O)OCCCCCCCCCCC)c1
- Structural formula attached as image file (if other than submission substance): see Fig. 3648-20-2.jpg

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River
- Justification: the rat was chosen because of its use as a predictor of reproductive toxic change in man and the
requirement for a rodent species by regulatory agencies. The CD strain was used because of the
historical control data available in this laboratory.
- Age at study initiation:10 - 11 weeks
- Weight at study initiation:220-267 g
- Housing: Mated females were singly housed in clear polycarbonate cages with stainless steel wire lids and virgin loose pulp as bedding
- Diet :ad libitum - pelleted diet (UAR Alimentation, Villemoisson, France)
- Water: ad libitum - filtered tap water
- Acclimation period: 1 - 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2 °C
- Humidity (%): 55% +/- 15%
- Photoperiod :12 hour cycle dark/light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Formulations were prapared on a weekly basis and stored at ambient temperature in the dark

VEHICLE
- Justification for use and choice of vehicle: Commonly used vehicle permitting dissultion/suspension of the test substance
- 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 body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability determined to be up to 2 weeks by analysis

Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- 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:
21 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg body weight
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg body weight
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg body weight
Basis:
actual ingested
No. of animals per sex per dose:
Three treatment groups of 21 - 22 females + a control group of 22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on preliminary study
- Rationale for animal assignment (if not random): Random by body weight stratification

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes, females were weighed and bodyweights reported for GD Days 0, 6, 9, 12, 15, 18 and 21.

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

AGD was also analysed used 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

Maternal developmental toxicity

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

Details on maternal toxic effects:
The incidence of general clinical signs, body weight changes and food consumption did not indicate any adverse effect of treatment.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
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:
No evidence of teratogenic effects was observed. 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.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

MATERNAL RESPONSE DATA:

All pregnant animals survived and no treatment-related clinical signs were noted.

There were no statistically significant changes in mean maternal body weights and body weight gains.

Food consumption was unaffected by treatment.

Maternal findings 

 

Treatment (mg/kg bw/day

 

0

250

500

1000

No. treated

22

21

21

22

% Gravid

100.0

100.0

100.0

90.9

% Mortality

0.0

0.0

0.0

0.0

Body weight (g)

 

 

 

 

GD 0

230 ± 13

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

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

 

 

 

 

 

Gravid uterine weight (g)

107 ± 13

98 ± 23

96 ± 29

103 ± 15

 

 

LITTER RESPONSE DATA:

No significant differences were observed in the number of corpora lutea or incidence of pre-implantation loss. The number of implants was slightly, but statistically significantly, lower than control at the low (250 mg/kg bw) and mid (500 mg/kg bw) doses. Post-implantation loss, number of resorptions, number of live foetuses, foetal sex ratio and foetal body weights were unaffected by treatment.

 

Male anogenital distance (AGD) was identical to control at 250 mg/kg bw but was slightly (3 - 4%) reduced at 500 and 1000 mg/kg bw. A statistically significant difference was apparent at 500 mg/kg bw after adjustment with the cubic root of foetal weight or when foetal body weight was used as covariate (with litter based analysis or mixed-effects model).

Litter data 

 

Treatment (mg/kg bw/day

 

0

250

500

1000

No. litters

22

21

21

20

No. Corpora lutea

15.7 ± 1.5

14.7 ± 2.0

14.9 ± 1.8

15.3 ± 1.7

% Pre-implantation loss/litter

3.0 ± 4.8

10.4 ± 17.7

8.4 ± 13.7

5.9 ± 10.2

No. implantation sites/litter

15.2 ± 1.8

13.2 ± 3.3 *

13.4 ± 2.9 *

14.3 ± 1.7

% Post-implantation loss/litter

6.4 ± 10.8

2.5 ± 5.9

8.0 ± 21.4

14.3 ± 1.7

No. Litters with dead foetuses

1

1

1

0

% Dead foetuses/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/litter

6.1 ± 10.7

2.2 ± 4.4

7.7 ± 21.5

3.7 ± 7.2

 

 

 

 

 

No. live litters

22

21

20

20

No. Live foetuses/litter

14.2 ± 1.9

12.9 ± 3.3

13.2 ± 2.7

13.8 ± 2.0

Sex ratio (% males/litter)

48.4 ± 12.1

51.1 ± 19.5

42.9 ± 13.7

49.9 ± 15.8

Foetal body weight (g)

 

 

 

 

Males

5.69 ± 0.31

5.75 ± 0.34

5.78 ± 0.25

5.69 ± 0.30

Females

5.37 ± 0.32

5.37 ± 0.39

5.48 ± 0.19

5.39 ± 0.28

Ano-genital distance (mm)

 

 

 

 

No litters examined

18

17

16

16

Males

2.96 ± 0.12

2.95 ± 0.15

2.85 ± 0.11

2.86 ± 0.15

Females

1.04 ± 0.06

1.06 ± 0.06

1.07 ± 0.06

1.09 ± 0.06

Ano-genital distance/body weight

 

 

 

 

Males

1.65 ± 0.08

1.65 ± 0.08

1.59 ± 0.05 *

1.60 ± 0.09

Females

0.59 ± 0.03

0.60 ± 0.03

0.61 ± 0.03

0.62 ± 0.04

 * Significantly different from controlp< 0.05

 

Malformations occurred in one foetus at 250 mg/kg bw (omphalocele) and in one foetus at 1000 mg/kg bw (diaphragmatic hernia), these isolated cases being considered incidental. Common external (club foot) and visceral (left umbilical artery) variations were seen in individual foetuses, with no indication of a treatment related adverse effect.

 

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

 

An increased occurrence of lumbar ribs was observed in foetuses from the 500 and 1000 mg/kg bw treatment groups, relative to controls. The mean percentage of affected foetuses per litter amounted to 10.3% in controls, 20.8% at 250 mg/kg bw, 46.6% at 500 mg/kg bw and 25.4% at 1000 mg/kg bw. The historical control range was from 6.8% to 19.4%. The incidence of affected litters was also significantly increased at 500 mg/kg bw. Long supernumerary ribs were observed in one foetus at 250 mg/kg bw and in one foetus treated at 1000 mg/kg bw. Supernumerary 14th ribs showed pinpoint ossification sites or were short in both 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 treated groups compared to controls was not considered toxicologically meaningful. All foetuses had 26 presacral vertebrae.

Foetal malformations/variations 

 

Treatment (mg/kg bw/day

 

0

250

500

1000

No. fetuses (litters) examined

 

 

 

 

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)

14thrib(s), supernumerary (any)

17 (10)

29 (13)

60 ** (17 *)

32 * (12)

Long

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

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**

 * Significantly different from controlp< 0.05                      ** Significantly different from controlp< 0.01

Applicant's summary and conclusion

Conclusions:
Developmental toxicity has been investigated using methods equivalent to OECD Test Guideline No. 414. 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. 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 No Observed Adverse Effect Level (NOAEL) is therefore regarded as being 1000 mg/kg bw/day, the highest dose investigated.
Executive summary:

Developmental toxicity has been investigated using methods equivalent to OECD Test Guideline No. 414. 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 No Observed Adverse Effect Level (NOAEL) is therefore regarded as being 1000 mg/kg bw/day, the highest dose investigated.