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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:
other: 1a GLP, OECD 414 Guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test compound: dimethylsulfoxide
CAS no.: 67-68-5
Source: Elf Aquitaine Production
Batch number: T8315
Purity: 99.89%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
ANIMALS
- Strain : Crl CD (SD) BR
- Breeder: Crl CD (SD) BR, from Charles River (Saint-Aubin-lès-Elbeuf, France),
- Age at the beginning of the treatment period: 10 weeks old
- Weight at the beginning of the treatment period: mean body weight of 254 g (range: 223 g to 289 g).
- Acclimation: 5-days before the beginning of the treatment period
- Food: Maintenance pelleted diet, type A04C (UAR, Villemoisson-sur-Orge, France) ad libitum
- Water: filtered (0.22 µm filter) tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Housing: individually in polycarbonate cages (43.0 x 21.5 x 20.0 cm) and each cage contained autoclaved sawdust
- Temperature: 21 + 2°C
- Relative Humidity: 50 + 20%
- Light/dark cycle : 12 h/12 h (07:00 - 19:00)
- Ventilation : about 8 to 10 cycles/hour of filtered, non-recycled air.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The dosage forms were prepared for up to seven days of treatment in advance, according to previously demonstrated stability and were stored refrigerated pending daily delivery.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Chemical analysis of preparations
Since the preparations were solutions, no homogeneity contol was required. Before the start of treatment, the suitability of the proposed preparation procedure was determined by the analysis of the stability of solutions prepared under the same conditions. During the course of the treatment period, a check of the concentration was performed on solutions prepared for use on the study.

- Stability
Two preparations were made, under the conditions representative of those of the study (if possible, on a typical quantity for one-day's treatment):
. a preparation at the lowest concentration (20 mg/ml),
. a preparation at the highest concentration (1000 mg/ml).
These solutions were stored at +4 °C and sampled on days 0, 4 and 9 storage.
The samples were stored frozen at -20 °C pending analysis (day 9 included) when all samples were assayed.

- Achieved concentration
The concentration of the samples of each solution (including the control) prepared for use on the first day of treatment of the first mated females and on the last day of treatment of the last mated females was determined.
Details on mating procedure:
Day 0 of pregnancy was designated as the day of confirmed mating.
Day 0 post-coitum (p.c.) designated the day when vaginal plug was observed.
Duration of treatment / exposure:
10 days, days 6-15 of gestation
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 200, 1000, 5000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Details on study design:
The dose-levels were selected on the basis of a dose-range finding study conducted at 200, 5000 and 10000 mg/kg/day. The 5000 mg/kg/day dose-level was slightly maternotoxic and the 10000 mg/kg/day was moderately maternotoxic. Hence, 5000 mg/kg/day was considered to be a suitable high dose-level for the present study.

Examinations

Maternal examinations:
Clinical signs including mortality and evidence of abortion were checked daily.
Food consumption and body weight were recorded at designated intervals during pregnancy.
On day 20 of pregnancy, females were killed. The gravid uterus was weighed and fetuses removed by hysterectormy. Females were examined macroscopically.
Ovaries and uterine content:
On day 20 p.c., the animals were humanely killed and examined macroscopically. The gravid uterus was weighed to allow calculation of the maternal net body weight change. The ovaries and uterine contents were examined and the following litter parameters numbered: corpora lutea, implantation sites, live and dead fetuses, early and late resorptions or scars.
Fetal examinations:
The fetuses were weighed, sexed and evaluated for external abnormalities.
The soft tissues were examined in half of the rat fetuses by serial sectioning after fixation in Bouin’s fluid (Wilson, 1965). In addition, kidneys of the fetuses presented renal alterations were examined microscopically. All the rabbit fetuses from the first 20 litters were evaluated by fresh dissection.
The skeleton was examined in half of the rat fetuses after alizarin red staining (Dawson, 1926); all the rabbit fetuses were examined after staining of both bone and cartilage using alizarin red and alcian blue (Peters, 1977).
The fetal findings were described according to the glossary of the International Federation of Teratology Societies (Chahoud et al., 1999) and classified as malformations or variations (Wise, 1997).
Statistics:
Group mean values of each treated group were compared to control group mean values by one-way analysis of variance and Dunnett test (Dunnett, 1955, 1964), mean values being considered as normally distributed and variances as homogeneous.
Proportions were compared by Fisher exact test (Fisher, 1934).
A difference was statistically significant at p<0.05.
For litter and fetal findings, the experimental unit of comparison was the litter

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
There were no clinical signs observed in treated or control groups. No maternal deaths or abortions occurred in any group.
Lower food consumption (-11% compared to control) was noted in females of the 5000 mg/kg group during the treatment period.
The body weight gain of the gravid females given 5000 mg/kg/d was slightly lower that of controls, during the treatment period: 41 g vs. 60 g (i.e. -32%, p<0.001); this resulted in mean absolute body weight on day 20 of pregnancy lower by -4% (365g vs. 382g) compared to controls) (Table 1).
No macroscopic findings were noted at necropsy in any of the females.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 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:no effects

Details on embryotoxic / teratogenic effects:
Pre- and post-implantation losses were similar in all groups. No treatment-related effects were observed on the number of foetuses or the sex ratio.
In the 5000 mg/kg/day group, foetal body weights 5table 2) were slightly lower than that of controls: 3.54 g vs. 3.79g (-7%, p< 0.05), an indirect consequence, at least in part, of decreased maternal food consumption and body weight gain (Table 1).
No external malformations or anomalies were observed in foetuses from any group. The soft tissue variations were confined to a slightly, not statistically significant, increased incidence of dilated renal pelvis (Table 3) only statistically significant at 200 mg/kg bw/d, associated at 5000 mg/kg bw/d with a slightly increased incidence of dilated ureter(s). In both cases, the incidences were in the range of the historical control values. At microscopic examination, the foetal kidneys displaying dilatation of the renal pelvis had the same morphological structure in the control and in the treated groups and the contralateral kidney (in the case of unilateral change) had the same structure as the affected kidney.
No treatment-related soft tissue malformations were observed. There were no treatment-related skeletal variations or malformations in any group. An increased incidence of reduced or delayed ossification of ribs was observed in foetuses of the 5000 mg/kg group Table 4). This skeletal anomaly is considered to be a consequence of the lowered foetal body weights observed for this group.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean values of food consumption, body weight gain and body weight in pregnant females (in grams)

Dose-level (mg/kg/day)

0 (Control)

200

1000

5000

Number of pregnant females

18

22

22

23

Food consumption, day 6 to day 15 of pregnancy

28+2

27+3

28+2

25+3***

Body weight gain, day 6 to day 15 of pregnancy

60+8

59+10

56+9

41+14***

Body weight at termination (day 20 of pregnancy)

382+25

384+30

380+26

365+36

Net body weight gain (from day 6 of pregnancy)

51.5+13.0

51.2+16.0

54.0+7.8

42.2+14.9

Values are Mean+Standard Deviation

Except where stated: no statistical significance

*** : p < 0.001, ANOVA + Dunnett test

Table 2: Mean values of litter data

Dose-level (mg/kg/day)

0 (Control)

200

1000

5000

Number of pregnant females

18

22

22

23

Corpora lutea per dam

19.1+3.1

17.2+2.4

16.6+3.6

17.9+2.5

Implantation sites per dam

13.8+2.9

14.3+2.5

12.9+4.0

13.0+3.4

Live fetuses per dam

13.2+3.1

13.8+2.7 

12.5+4.2

12.4+3.9

Dead fetuses per dam

0.0

0.0

0.0

0.0

Resorptions (early + late) per dam

0.6+0.8

0.5+1.0

0.3+0.7

0.6+1.0

Post-implantation loss (% of implantation sites)

4.0

3.5

2.5

4.4

Fetal weight

Males

3.90+0.27

3.94+0.22

4.00+0.28

3.64+0.30**

Females

3.69+0.29

3.73+0.26

3.75+0.31

3.46+0.28*

Sex ratio (% of males) 

47.5

55.1

50.0

48.8

Except where specified, values are Mean+Standard Deviation

Except where stated: no statistical significance

* : p <.05; ** : p <0.01, ANOVA + Dunnett test

Table 3 . Incidence of dilated renal pelvis and ureter(s) in the OECD 414 study in rats

Dose-level (mg/kg/d)

0

200

1000

5000

 

 

 

 

 

# Foetuses evaluated

114

147

133

139

# Litters evaluated

18

22

21

22

Dilatation of pelvis of kidney(s)

 

 

 

 

# Foetuses affected (%) (a)

2 (1,8)

13 (8,8)

7 (5,3)

7 (5,0)

# Litters affected (%) (a)

1 (5,6)

7 (31,8)

6 (28,6)

6 (27,3)

Dilatation of ureter(s)

 

 

 

 

# Foetuses affected (%) (a)

3 (2,6)

8 (5,4)

2 (1,5)

10 (7,2)

# Litters affected (%) (a)

2 (11,1)

5 (22,7)

2 (9,5)

8 (36,4)

Both finding associated

 

 

 

 

# Foetuses affected

2

5

2

3

 

 

 

 

 

Except where stated: no statistical significance 

 

 

 

* : p 0.05

 

 

 

 

(a) Fisher exact test

 

 

 

 

 

Table 4: Summary of remarkable skeletal findings

Dose-level (mg/kg/day)                        0 (Control)                   200                              1000                            5000

---------------------------------------------------------------------------------------------------------------------------------------------------------

Fetuses evaluated                                 124                            156                              143                              146

Litters evaluated                                    18                                22                                22                                23

Reduced ossification of rib(s)

      . affected fetuses per litter (%) (a)  1.4 + 4.0              2.2 +7.3                      2.7 + 6.1                      11.5 + 16.3**

      . fetal incidence (%) (b)                1.6                               1.9                               3.5                               13.7***

      . litter incidence (%) (b)                   11.1                             9.1                               18.2                             39.1

----------------------------------------------------------------------------------------------------------------------------------------------------------

Except where stated: no statistical significance         ** : p 0.01     *** : p 0.001             (a) ANOVA + Dunnett test        (b) Fisher exact test

Applicant's summary and conclusion

Conclusions:
Under these experimental conditions, the No-Observed-Adverse-Effect-Level (NOAEL) was 1000 mg/kg/d for maternal toxicity. A NOAEL has been determined at 1000 mg/kg/d for foetal toxicity.
Executive summary:

The potential of Dimethylsulfoxide (DMSO) to induce developmental toxicity after maternal exposure during the critical period of organogenesis was evaluated in rat according to OECD Guideline N° 414 (12th May 1981) and US EPA, CFR title 40, part 798-4900 (1985) and in compliance with Good Laboratory Practices.

DMSO was administered orally by gavage to four groups of 25 bred female Sprague-Dawley rats once daily from gestation days 6 through 15 inclusive at dosage levels of 0, 200, 1000 and 5000 mg/kg/d.

Clinical signs and mortality were checked daily. Food consumption and body weight were recorded at designated intervals during pregnancy. On day 20 of pregnancy, females were sacrificed. The gravid uterus was weighted and foetuses were then removed by hysterectomy. Females were examined microscopically. Litter parameters were recorded: number of corpora lutea, implementation sites, early and late resorptions, dead and live foetuses. Foetuses were weighted, sexed and submitted to external examination and then to soft tissue and skeletal examination.

No clinical signs and no unscheduled deaths were observed in any group. No abortion occurred in any animal. The food consumption and body weight gain of the females given 5000 mg/kg/g were slightly lower than that of controls. No treatment related macroscopic findings were observed. 

The pre-and post implantation losses were similar in all groups. No treatment related effects were observed on the number of foetuses or the sex ration. In the 5000 mg/kg/d group, the body weight of the foetuses was slightly lower than that of controls (this was considered to be at least in part, an indirect consequence of the effect on the maternal body weight).

No treatment related external malformations or anomalies were noted in foetuses in any group. An increased incidence of two soft tissue anomalies was observed: dilated renal pelvis for the foetuses of all treated groups, associated at 5000 mg/kg/d with an increase incidence of dilated ureter(s). No treatment-related soft tissue or skeletal variations malformations were noted in any group. An increased incidence of reduced ossification of rib was observed in the foetuses of the 5000 mg/kg/d. These findings might be considered to be a delayed ossification, as a consequence of the lower body weight of the foetuses recorded at hysterectomy. No treatment related microscopic changes were noted in the kidneys of the foetuses presenting dilated renal pelvis at soft tissue examination. The observed dilated renal pelvis was therefore considered not to be an adverse effect and might be related to diuretic properties of DMSO.

Under these experimental conditions, the No-Observed-Adverse-Effect-Level (NOAEL) was 1000 mg/kg/d for maternal toxicity. A NOAEL has been determined at 1000 mg/kg/d for foetal toxicity.