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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
Study period:
August to November 1995
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted May 12, 1981
Deviations:
yes
Remarks:
room temperature of 20 +/- 2 °C instead of 22 +/- 2 °C; acclimation period of 3-5 days instead of at least 5 days; acclimation period after mating
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): WITAMOL 110/LINPLAST 610 P
- Substance type: pure active substance
- Physical state: liquid
- Lot/batch No.: 950608
- Expiration date of the lot/batch: no data
- Stability under test conditions: stable in vehicle over 8 days
- Storage condition of test material: ambient temperature, in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation: approx. 9 weeks
- Weight at study initiation: approx. 210 g
- Fasting period before study: no
- Housing: single in polypropylene cages with stainless steel grid bottoms and mesh tops
- Diet: Rat and Mouse Breeder Diet No. 3 (Expanded) SQC (by Special Diets Services (SDS) Limited, Stepfield, Witham, Essex, UK) ad libitum
- Water (e.g. ad libitum): domestics mains water adlibitum
- Acclimation period: 3-5 days prior dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 2
- Humidity (%): 50 +/- 15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: October 20, 1995 To: November 6-8, 1995

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
maize oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Mixing of requisite test material with correct quantity of vehicle in glass container by gentle manual inversion. The dose solutions were prepared freshly for the first 5 days of the dosing period. For the remaining 8 days of dosing, a single batch was prepared for each dose level, and a suitable aliquot from each batch was dispensed daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Amount of vehicle (if gavage): total volume applied: 5 ml/kg
- Lot/batch no. (if required): no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Triplicate samples were taken from all dosing solutions on 2 occasions, the first and fifth day of dosing period. These samples were analysed for concentration and homogeneity.
Details on mating procedure:
The female rats were delivered time-mated by the supplier. The delivery consisted of 3 subbatches mated over 3 successive days. On delivery, one batch was on day 1 of gestation, a second batch on day 2 and a third on day 3 of gestation (Day of mating = day 0 of gestation).
No more than 2 study females were inseminated by the same male.
Duration of treatment / exposure:
days 6 to 16 of gestation
Frequency of treatment:
daily
Duration of test:
until day 20 of gestation
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/d
Basis:
actual ingested
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, with particular attention being paid to the period 1-2h after dosing


BODY WEIGHT: Yes
- Time schedule for examinations: days 4, 6-17 and 20 of gestation


FOOD CONSUMPTION : Yes, from day 3 of gestation
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: macroscopic examination of thoracic and abdominal 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 resorptions: Yes
- Number of late resorptions: Yes
- Other: position of implantation sites

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
-Other: sex of foetuses, weight of live foetuses

Statistics:
Body weight gain:
Parametric analysis of variance [Snedecor, GW and Cochran, WG (1980) Statistical Methods, 7th Edition, 213-254 and 365-392, Iowa State University Press.] Pairwise comparisons between each treatment level and control using Dunnett's test [Dunnet, CW (1964) Biometrics, 20, 482-491.] Where there was significant heterogeneity of variance, a log or square root transformation was used in an attempt to stabilise the variances. Where transformation failed to stabilise the variances, the Kruskal-Wallis test was used instead [Hollander, M and Wolfe, DA (1973) Non-parametric Statistical Methods, John Wiley and Sons, 114-137.]

Other parameters:
interpretation based on inspection of the individual and group values (formal statistical analysis not considered useful to conduct).

Historical control data:
no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical observations:
Slight increase in the incidence of piloerection at 1000 mg/kg bw/d (see table 1)

Body weight performance:
At 1000 mg/kg bw/d slight increase in weight gain over days 6-17 of gestation, principally between days 13-17, with the increase over days 6-17 just failing to achieve statistical significance (P> 0.05). At 100 and 500 mg/kg/d, mean weight gains were marginally greater than control, but the differences were considered too small to be attributed to treatment. (See table 2)

Food consumption:
Slight increase in food consumption from day 10 of gestation in high dose group. In low and mid dose group food consumption was essentially similar to that of the controls. (See table 3)

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Foetal abnormalities and variants:
Increased incidences of foetuses with 14th ribs in mid and high dose groups; the incidence in low dose group was similar to control (see table 4).

Increased incidence of foetuses with retarded sternebrae in the high dose group compared with control. In the low and mid dose groups the incidences of retarded sternebrae were not noticeable different from Control. (See table 5)



Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Clinical Observations:

Table1:

Observation/finding

Dose level [mg/kg bw/d]

0

100

500

1000

Brown staining nasal region

1

1

0

2

Lower teeth cut; upper teeth broken

0

1

0

0

Wet/stained vagina

0

0

1

1

Soft faeces

0

0

0

1

Lump upper caudal region

1

0

0

0

Scabbing

1

1

0

0

Unkempt coat

0

0

1

0

Hairloss

0

0

0

1

Piloerection

0

1

2

5

Aggressive/agitated behaviour

0

0

1

1

Irregular respiration

0

0

0

1

Thin

0

1

0

0

Connected spleen and left kidney

1

0

0

0

Distended uterine horn

0

0

1

0

Body weight performance:

Table 2: Group mean body weight (g) ± Standard Deviation (pregnant animals only)

Day of Gestation

Dose level [mg/kg bw/d]

0

100

500

1000

4

225 +/- 10

227 +/- 11

224 +/-11

230 +/- 8

6

242 +/- 9

243 +/- 9

241 +/- 13

247 +/-9

9

259 +/- 11

262 +/- 10

260 +/- 15

263 +/- 9

13

294 +/- 13

297 +/- 12

295 +/- 18

299 +/-11

17

335 +/- 16

342 +/- 13

341 +/- 28

348 +/- 14

20

386 +/- 24

394 +/- 19

389 +/- 36

397 +/-18

Gain Days 6-17

93 +/- 11

99 +/- 10

99 +/- 19

102 +/-8

% of control

-

106

106

110

            Means are based on 17-25 animals

Food consumption:

Table 3: Group mean food consumption (g) ± Standard Deviation (pregnant animals only)

Day of Gestation

Dose level [mg/kg bw/d]

0

100

500

1000

3

28

28

28

28

4

26

27

25

27

5

28

28

28

29

6

25

24

25

26

7

28

28

29

29

8

28

28

29

29

9

30

29

30

31

10

30

32

32

33

11

32

32

32

35

12

31

32

32

34

13

31

31

31

34

14

33

33

34

36

15

35

35

34

36

16

34

34

35

36

17

38

37

36

39

18

34

36

35

37

19

28

29

29

31

Total Mean Consumed Days 6-16

337

338

343

359

% of Control

-

100

102

107

Means are based on 17-25 animals

Pregnancy performance:

Lower pregnancy frequencies in low and high dose groups compared to control and mid dose groups. These findings are considered incidental as events leading to the establishment of pregnancy should have occurred prior to commencement of treatment.

No indications of an effect of treatment in the frequency of embryo-foetal deaths or on foetal weight.

Foetal abnormalities and variants:

The incidence of major abnormalities did not indicate an effect of treatment. All abnormalities seen at the high dose have been seen previously in the test laboratory.

The incidence of minor visceral abnormalities was similar in all groups.

The incidence of skeletal abnormalities and variants, including the ossification parameters, other than mentioned in the section ¿Details on embryonic and teratogenic effects¿, were essentially similar in all groups.

Table 4: Group incidence of minor foetal abnormalities and variants

Abnormality/Variant

Dose level [mg/kg bw/d]

0

100

500

1000

Incidence of Foetuses (Litters)

Skeletal:

 

 

 

 

Sutural bone

1 (1)

2 (2)

1 (1)

1 (1)

Cervical rib(s)

0

3 (3)

1 (1)

4 (3)

Minimal distortion of rib(s)

0

1 (1)

1 (1)

1 (1)

Misshapen 6th sternebra

1 (1)

1 (1)

1 (1)

0

Asymmetric pelvic girdle

0

0

0

1 (1)

Number of ribs:

 

 

 

 

Reduced 13th rib

0

0

0

1 (1)

13 complete rib(s)

142 (25)

95 (17)

97 (22)

57 (17)

14th vestigial supernummerary rib(s)

12 (9)

14 (8)

37 (15)

55 (15)

Number with minor skeletal abnormality/variant

2 (2)

5 (5)

4 (4)

7 (5)

Total number examined skeletally

154 (25)

109 (17)

134 (22)

113 (19)

Table 5: Group incidence of incomplete ossification parameters

Parameter

Dose level [mg/kg bw/d]

0

100

500

1000

Incidence of Foetuses (Litters)

Incomplete ossification affecting:

 

 

 

 

¿3 skull bones

14 (8)

8 (6)

21 (11)

20 (11)

¿ 4 skull bones

3 (3)

1 (1)

5 (5)

4 (2)

Thoracic vertebral centrum(a)

4 (3)

6 (4)

3 (2)

4 (3)

2nd and 4th metacarpal(s)

1 (1)

0

0

1 (1)

Pubis(es)

1 (1)

1 (1)

0

1 (1)

Ischium

1 (1)

0

0

1 (1)

Cervical vertebral arch(es)

0

0

1 (1)

0

Lumbar vertebral centrum(a)

0

0

0

1 (1)

Sacral vertebral arch(es)

12 (8)

5 (3)

3 (2)

2 (2)

2nd and 4th metatasal(s)

0

0

1 (1)

0

Unossified 5th metacarpal(s)/metatarsal(s)

30 (16)

26 (11)

17 (7)

32 (14)

Number of sternebrae retarded:

 

 

 

0

103 (24)

72 (16)

70 (18)

35 (15)

1

33 (16)

29 (11)

42 (16)

51 (17)

2

17 (9)

8 (5)

20 (9)

20 (11)

3

1 (1)

0

0

2 (1)

>3

0

0

1 (1)

4 (2)

Total number examined

154 (25)

109 (17)

134 (23)

113 (19)

Applicant's summary and conclusion

Conclusions:
The structurally related substance 1,2-Benzenedicarboxylic acid, di-C6-10-alkyl esters caused slight maternal effects only at 1000 mg/kg bw/d indicated by slightly increased body weight gain and food consumption.
Foetal effects were confined to increases in the incidence of vestigial supernumerary ribs at 500 and 1000 mg/kg bw/d and to a slight increase in the incidence of retarded sternebrae at 1000 mg/kg bw/d. Both of these findings were considered to be of a minor nature.
Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 500 mg/kg bw/d. The No Observed Adverse Effect Level (NOAEL) for developmental toxicity was considered to be 1000 mg/kg bw/d.
Executive summary:

A study of developmental toxicity revealed slight maternal effects only at 1000 mg/kg bw/d of the structurally related substance 1,2-Benzenedicarboxylic acid, di-C6-10-alkyl esters indicated by slightly increased body weight gain and food consumption. Foetal effects were confined to increases in the incidence of vestigial supernumerary ribs at 500 and 1000 mg/kg bw/d and to a slight increase in the incidence of retarded sternebrae at 1000 mg/kg bw/d. Both of these findings were considered to be of a minor nature. The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 500 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) for developmental toxicity was considered to be 1000 mg/kg bw/day.