Dimethyl sulfoxide

Developmental toxicity / teratogenicity

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:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl sulfoxide

EC Number:

200-664-3

EC Name:

Dimethyl sulfoxide

Cas Number:

67-68-5

Molecular formula:

C2H6OS

IUPAC Name:

dimethyl sulfoxide

Details on test material:

Test compound: dimethylsulfoxide
CAS no.: 67-68-5
Source: Aldrich
Batch number: 35345
Purity: 99.98%

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

ANIMALS
- Strain : KBL New Zealand White rabbits
- Breeder: Charles River (Châtillon-sur-Chalaronne, France)
- Age at the beginning of the treatment period: 18-20 weeks old
- Weight at the beginning of the treatment period: mean body weight of 3430 g (range: 2905 g to 4260 g).
- Acclimation: 6-days before the beginning of the treatment period
- Food: Maintenance pelleted diet, A110C (UAR, Villemoisson-sur-Orge, France)ad libitum
- Water: filtered (0.22 µm filter) tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Housing: individually in stainless steel cages (64.0 cm x 48.0 cm x 41.0 cm)
- 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 test item was administered as a solution in the vehicle. The test item was dissolved in the required quantity of vehicle in order to achieve the concentrations of 20, 60 and 200 mg/mL and then homogenized using a magnetic stirrer. The test item dosage forms were made at for up to 9 days of treatment (stability data available from previous studies) and were stored at +4°C prior to use.
The dosage forms were delivered each day to the animal room.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Chemical analysis of the dosage forms
The concentration of samples taken from each dosage form (including the control) prepared for use on the first day of treatment and the last day of treatment was determined.

Details on mating procedure:

Females were mated at the breeder's facilities. The day of confirmed mating (visual assessment) was designated as day 0 post-coitum.

Duration of treatment / exposure:

day 7 to day 28 post coitum, inclusive

Frequency of treatment:

daily

Duration of test:

Sacrifice on day 29 post coitum

No. of animals per sex per dose:

24 mated females per group

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, 1000 and 5000 mg/kg/day. There was no maternotoxicity or adverse effects on the embryofetal development at 200 and 1000 mg/kg/day, whereas the 5000 mg/kg/day dose-level was severely maternotoxic. Hence, 1000 mg/kg/day was considered to be a suitable high dose-level for the present study.

Examinations

Maternal examinations:

- Morbidity and mortality
Each animal was checked for mortality or signs of morbidity:
. at least twice a day during treatment period,
. at least once a day on other days.
- Clinical signs
Each animal was observed at least once a day, at approximately the same time for the recording of clinical signs (including evidence of abortion/resorption for the females).
- Body weight
The body weight of each female was recorded on days 2, 4, 6, 7, 9, 12, 15, 19, 24 and 29 post-coitum.
The net body weight was calculated (gross body weight minus gravid uterine weight), as well as the gross and net body weight changes.
- Food consumption
The quantity of food consumed by each female was recorded for the following intervals: 2-4, 4-6, 6-7, 7-9, 9-12, 12-15, 15-19, 19-24 and 24-29 post-coitum. Any obvious spillage of food was documented.
Food intake per animal and per day was calculated.
- Water consumption
The quantity of water consumed by each female was recorded for the following intervals: days 2-4, 4-6, 6-7, 7-9, 9-12, 12-15, 15-19, 19-24 and 24-29 post-coitum.
- Macroscopic post-mortem examination
All females were submitted to a macroscopic post-mortem examination of the principal thoracic and abdominal organs.
A gross evaluation of placentas was also undertaken.
The number of corpora lutea and implantation sites was recorded whenever possible in the females that died.

Ovaries and uterine content:

- Hysterectomies
On day 29 post-coitum, all the females were killed by an intravenous injection of thiopental sodium.
The weight of the gravid uterus was recorded for each pregnant female at hysterectomy (with at least one live fetus).
The ovaries and uterus of the females were examined to determine:
. number of corpora lutea,
. number and distribution of dead and live fetuses,
. number and distribution of early and late resorptions,
. number and distribution of implantation sites (or uterine scars).
Early resorptions refer to evidence of implantation without recognizable embryo or fetus; late resorptions refer to dead embryo or fetus with external degenerative changes; scars refer to evidence of implantation without recognizable structure (embryo, fetus or placenta).

Fetal examinations:

These examinations were carried out only in the first 20 litters (females with at least one live fetus). The other litters were discarded without further investigation.
The photographs of the fetuses were taken to document findings and kept with the study archives.
The findings were described according to the glossary of the International Federation of Teratology Societies (IFTS) and classified as malformation or variation:
. malformation refers to a permanent structural change that is likely to adversely affect the survival or health,
. variation refers to a change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health (this might include a delay in growth or morphogenesis that has otherwise followed a normal pattern of development).

- Body weight
The body weight of each live fetus was recorded.
- External examination
Each fetus was submitted to a detailed external examination, which included the observation of all visible structures, surfaces or orifices. Dead fetuses were then discarded. The live fetuses were killed by a subcutaneous injection of thiopental sodium.
- Soft tissue examination
. Body
All live fetuses were subjected to a detailed fresh dissection of the soft tissue, which included the observation of all the organs and structures of the head, neck, thorax and abdomen. The kidneys of all fetuses were sampled and preserved in 10% buffered formalin. No microscopic examination was deemed necessary.
. Head and brain
The heads of half of the fetuses were removed and processed (after fixation in Harrisson’s fluid) for evaluation of brain, nasal passages and tongue (and other structures, where appropriate). The brain of the other half of the fetuses was sampled and fixed in Bouin's fluid. It was then horizontally sectioned and examined.
. Skeletal and cartilage examinations
The carcasses of the fetuses were fixed with ethyl alcohol. A detailed examination of the skeleton was performed after staining with alizarin red S and alcian blue. This examination included the observation of all the bone and cartilage structures of the head, spine, rib cage, pelvis and limbs.
- Sex of fetuses
The sex of each live fetus was determined at the time of the dissection.
The sex of each dead fetus was determined at the time of the hysterectomy.

Statistics:

Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Percentage values were compared by the Fisher exact probability test.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
There was no mortality attributed to treatment with the test item.
There were no clinical signs of toxicity in any group.
The body weight, food consumption and water consumption were not affected by the treatment at 100 mg/kg/day.
In the 300 mg/kg/day group, there was only a slight reduction of food consumption during the first 2 days of treatment.
At 1000 mg/kg/day, there was a moderate reduction of food consumption and body weight gain, during the first part of the treatment period (days 7-15 of pregnancy).

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

2

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No macroscopic findings were observed at any dose-level, which were ascribed to treatment with the test item.
No treatment-related effects were observed on the pre- or post-implantation loss, the foetal weight or the sex ratio.

Foetal examinations
No malformations or variations were noted at external, soft tissue or skeletal examination that were ascribed to treatment with the test item or considered to be of toxicological significance.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Basis for effect level:

fetal/pup body weight changes

external malformations

skeletal malformations

visceral malformations

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

The mean body weight gain of the pregnant animals is summarized as follows: Mean body weight gain (g)

Dose-level (mg/kg/day)	0	100	300	1000
Whole treatment period: D7-D29	294	341	354	270
First days of treatment: D7-D9	43	16	18	-25#
First part of treatment: D7-15	75	113	96	47
Second part of treatment: D15-D29	194	230	58	224
Net body weight change	-230	-183	-139	-256
#: p0.001

The mean food consumption of the pregnant animals is summarized as follows: Mean food consumption (g/day)

Dose-level (mg/kg/day)	0	100	300	1000
Whole treatment period: D7-D29	143	142	144	130
First days of treatment: D7-D9	179	162	147**	122#
First part of treatment: D7-15	153	156	145	127*
Second part of treatment: D15-D29	129	136	144	133
*: p0.05, **: p0.01, #: p0.001

 

Mean values of litter data

Dose-level (mg/kg/day)                           0 (Control)                   100                              300                              1000

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Number of pregnant females               22                                21                                24                                24

Corpora lutea per doe                          11.9 + 2.0                    11.6 +2.8                    11.7 + 2.6                    11.6 + 2.5

Implantation sites per doe                  10.3 + 2.1                    10.3 +3.4                    9.3 + 3.3                      10.1 + 2.4

Live fetuses per doe                 9.7 + 1.9                      9.8 +3.0                      9.1 + 3.2                      9.4 + 2.3

Dead fetuses per doe                             0.0                               0.0                               0.0                               0.0

Resorptions (early + late) per doe        0.5 + 1.0                      0.5 +0.7                      0.3 + 0.5                      0.7 + 0.9

Post-implantation loss (% of implantation sites)   

                              5.8                               5.5                               2.7                               7.0

Fetal weight            . Males                      37.0 + 4.1                    37.6 +7.8                    38.1 + 5.7                    36.3 + 3.9

                              . Females                     36.9 + 5.1                    35.5 +5.6                    35.3 + 4.7                    36.1 + 3.2

Sex ratio (% of males)                              53.3                             47.2                             47.8                             47.3    

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Except where specified, values are Mean + Standard Deviation                   No statistical significance was recorded

Applicant's summary and conclusion

Conclusions:

DIMETHYL SULFOXIDE, when administered daily to pregnant rabbit by gavage from day 7 to day 28 post-coitum at 100, 300 or 1000 mg/kg/day produced no signs of maternal toxicity at 100 mg/kg/day and induced a slightly decrease of the body weight gain during the first 2 days of treatment at 300 and 1000 mg/kg/day.
The embryofetal development was not affected and there were no teratogenic effects at any dose-level. Consequently, the No Effect Level for adverse effects on the embryofetal development was established at 1000 mg/kg/day.

Executive summary:

The potential of Dimethylsulfoxide (DMSO) to induce developmental toxicity after maternal exposure during the critical period of organogenesis was evaluated in rabbit according to OECD Guideline N° 414 (22nd January 2001) and US EPA, Health effects Test Guidelines OPPTS 870.3700, (August 1998) and in compliance with Good Laboratory Practices. DMSO was administered orally by gavage to four groups of 24 bred female KBL New Zealand White rabbits once daily from gestation days 7 through 28 inclusive at dosage levels of 0, 100, 300 and 1000 mg/kg/d. Clinical signs and mortality were checked daily. Body weight, food consumption and water consumption were recorded at designated intervals. On day 29post-coitum, the does were sacrificed and subjected to macroscopic examination. The gravid uterus was weighed to allow calculation of the net body weight change. The fetuses were removed by hysterectomy. The litter parameters were recorded, namely: number of corpora lutea, implantation sites, early and late resorptions, dead and live fetuses. The fetuses were weighed, sexed and submitted to external examination. A detailed examination of the soft tissue was performed by fresh dissection. Then the fetuses were submitted to a detailed examination of the skeleton (bone and cartilage) following alizarin/alcian blue staining.

There was no mortality attributed to treatment with the test item. There were no clinical signs of toxicity in any group. The body weight, food consumption and water consumption were not affected by the treatment at 100 mg/kg/day. In the 300 mg/kg/day group, there was only a slight reduction of food consumption during the first 2 days of treatment. At 1000 mg/kg/day, there was a moderate reduction of food consumption during the first part of the treatment period (days 7-15 of pregnancy) and body weight gain during the first 2 days of the treatment period (days 7 -9 of pregnancy). No macroscopic findings were observed at any dose-level that were ascribed to treatment with DMSO. No treatment-related effects were observed on the pre- or post-implantation loss, the foetal weight or the sex ratio. No malformations or variations were noted at external, soft tissue or skeletal examination that were ascribed to treatment with the test item or considered to be of toxicological significance.

Under these experimental conditions, DMSO administered daily to pregnant rabbit by gavage from day 7 to day 28 post-coitum at 100, 300 or 1000 mg/kg/day produced no signs of maternal toxicity at 100 mg/kg/day and was minimally to slightly maternotoxic at 300 and 1000 mg/kg/day. The embryofetal development was not affected and there were no teratogenic effects at any dose-level. The No Adverse Effect Level (NOAEL) for embryofetal development and maternal toxicity was established at 1000 mg/kg/day. The No Effect Level (NOEL) for maternal toxicity was established at 300 mg/kg/day.