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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 September 2006 - 23 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test article name: Dimethyl sulfoxide
CAS no. : 67-68-5
Source: ARKEMA France
Batch no. : 28/08/06
Purity: 99.977%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
ANIMALS
- Strain : Sprague-Dawley Crl CD (SD) IGS BR
- Breeder: Charles River Laboratories France, L'Arbresle, France
- Age at the beginning of the treatment period: 10 weeks old
- Weight at the beginning of the treatment period: 372 g (range: 333 g to 396 g) for the males and 241 g (range: 211 g to 280 g) for the females
- Acclimation: 6-days before the beginning of the treatment period
- Housing: the males were individually housed, except during pairing, in wire-mesh cages (43.0 x 21.5 x 18.0 cm). The females were individually housed, except during pairing and lactation, in polycarbonate cages (43.0 x 21.5 x 20.0 cm) containing autoclaved sawdust. Autoclaved wood shavings were provided as nesting material, a few days before delivery and during the lactation period.
- Food: SSNIFF R/M-H pelleted maintenance diet ad libitum
- Water: filtered (0.22 µm filter) tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature : 22 ± 2°C
- Relative humidity : 50 ± 20%
- Light/dark cycle : 12h/12h (7:00 - 19:00)
- Ventilation : about 12 cycles/hour of filtered, non-recycled air.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- Dosage form preparation: The test item was administered as a solution in the vehicle. The test item was mixed with 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 prepared for up to 9 days use (based on available stability information (stability for up to 9 days at +4°C, under nitrogen atmosphere), and were stored in glass bottles at +4°C and under nitrogen atmosphere until treatment.
- Treatment volume: 5 ml/kg
Details on mating procedure:
one female was placed with one male from the same dose-level group.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test item was determined by Gas Liquid Chromatography with Flame Ionisation Detection in samples taken from each dosage form (including the control) prepared for use in weeks 1, 4 and 9.
Duration of treatment / exposure:
- in the males: 15 days before mating, during the mating period (2 weeks) and until sacrifice (i.e. at least 4 weeks in total),
- in the females: 15 days before mating, during the mating period (2 weeks), during pregnancy and during lactation until day 21 post-partum inclusive.
Frequency of treatment:
7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CLINICAL EXAMINATIONS

- Morbidity and mortality: at least twice a day

- Clinical signs: at least once a day

- Body weight:

. Males: on day 1, then once a week until sacrifice

. Females: on day 1, then once a week until mated, then on days 0, 7, 14 and 20 pc and on days 1 and 4 pp and then weekly until day 21 pp.

- Food consumption

. Males: once a week (except during the mating period) until sacrifice

. Females: once a week during the pre-mating period and then on the following intervals: days 0-7, 7-14, 14-20 pc, and days 1-7, 7-14 and 14-21 pp.

PARTURITION

Females were allowed to drop their litters normally and rear their progeny until sacrifice.
Oestrous cyclicity (parental animals):
The oestrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the mating period, until the females mated.
Litter observations:
- Litter size: total litter size and numbers of pups of each sex were recorded as soon as possible after birth. The litters were observed daily.

- Clinical signs: daily

- Body weight: days 1 and 4 pp and then weekly until day 21 pp
Postmortem examinations (parental animals):
- Sacrifice
. Males: after the end of the mating period,
. Females: on day 22 pp,
. Females which had not delivered by day 25 pc: on day 25 pc
. Females which did not mate: 24 days after the end of the mating period.

- Organ weights: Epididymides, kidneys, Liver, Prostate, Seminal vesicles and Testes

- Macroscopic post-mortem examination: on all parent animals. In all females, the number of implantation sites and corpora lutea was recorded.

- Preservation of tissues: ovaries, prostate, seminal vesicles, uterus (horns and cervix), vagina, liver and kidneys, in 10% buffered formalin. Testes and epididymides, in Bouin's fluid

-Microscopic examination: ovaries, testes and epididymides of all males and females in the control and high-dose groups.
Postmortem examinations (offspring):
- Sacrifice
. Pups: on day 22 pp, gross external examination before sacrifice
Statistics:
- Data other than organ weights
Mean values were compared by one-way variance analysis and Dunnett test, (mean values being considered as normally distributed, variances being considered as homogeneous). Percentage values were compared by Fisher exact probability test.
- Organ weights
See attached document

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
All treated female groups had slightly lower body weight gains than the controls during the pre-pairing period but there were no effects during gestation or lactation and no effects on food consumption.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
All treated female groups had slightly lower body weight gains than the controls during the pre-pairing period but there were no effects during gestation or lactation and no effects on food consumption.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Minor differences at 1000 mg/kg/d, considered to be of no toxicological significance
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CHEMICAL ANALYSES OF THE DOSAGE FORMS
- Concentration: satisfactory agreement (± 8%) between the nominal and actual concentrations

CLINICAL EXAMINATIONS
- Mortality
. Males: no deaths in any group.
. Females: no deaths in any group.
One female treated at 300 mg/kg/day was sacrificed after the end of the pairing period as no evidence of mating has been observed.
Two mated females, one treated at 300 mg/kg/day and one treated at 1000 mg/kg/day were sacrificed after no delivery.

- Clinical symptoms
No treatment-related clinical signs were observed during the study in males and females.

- Body weight
. Males: no effect related to treatment
. Females: at all dose-levels, they gained slightly less weight (-11%, -26%, -22%) during the pre-pairing period than the controls, although weight gains during gestation and lactation were similar to the controls (see table 1 below).

- Food consumption: no change in food consumption


MATING AND FERTILITY DATA
- Mating index: no treatment-related effect
- Fertility index: no treatment-related effect
- Duration of gestation: no treatment-related effect
- Delivery data: no treatment-related effect

PATHOLOGY
- Organ weights
Minor differences, considered to be of no toxicological significance, were observed in the weights of liver between males given 1000 mg/kg/day and controls and of prostate and testis (see table 2 below).
- Macroscopic post-mortem examination:
No treatment-related effect
- Microscopic examination:
. Testicular Staging: No treatment-related changes were observed.
. Ovaries: No treatment-related changes were observed.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
>= 1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: marginally increased liver weights at 1000 mg/kg/day without macroscopic findings
Dose descriptor:
NOEL
Remarks:
reproductive performance (mating and fertility)
Effect level:
>= 1 000 mg/kg bw/day
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

- Mortality:
At 100 mg/kg/day, the number of pups dying between days 1 and 4 post-partum was statistically significantly higher than control values. The dead pups were distributed among six litters although as the number of dead pups in the groups treated at 300 or 1000 mg/kg/day was not significantly increased and as the viability index was still 93% and as there were no more deaths after day 4 post-partum, this was considered not to be related to treatment.

- Clinical signs:
No treatment-related effect

- Pup body weight:
No treatment-related effect

- Sex ratio:
No treatment-related effect

PATHOLOGY
Pup necropsy findings were limited to a highest incidence of pups with dilated renal pelvis in the group treated at 1000 mg/kg/day (see table 3 below). However, this effect was not statistically significant, in the range of the historical values (see table 4 below) and there was no dose-relationship between the groups treated at 100 or 300 mg/kg/d.
 
 

Effect levels (F1)

Dose descriptor:
NOEL
Remarks:
effects on the progeny
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Body weight

The main differences in mean body weight gain (in grams) are summarized as follows:

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Pre-mating

 

 

 

 

 

 

 

 

Days 1-15

+73

+75

(+3%)

+77

(+5%)

+75

(+3%)

+27

+24

(-11%)

+20

(-26%)

+21

(-22%)

Gestation

 

 

 

 

 

 

 

 

Days 0-20p.c.

/

/

/

/

+157

+154

(-2%)

+159

(+1%)

+149

(-5%)

Lactation

 

 

 

 

 

 

 

 

Days 1-21p.p.

/

/

/

/

+21

+27

(+29%)

+29

(+38%)

+25

(+19%)

/: not recorded,p.c.:post-coitum, p.p:post-partum.

 

Table 2: Organ weights

The principal differences (expressed in %) noted between test item-treated and control animals for the absolute and relative organ weights are given in the table below:

 

Sex

Male 

Female 

Group

2

3

4

2

3

4

Dose-level (mg/kg/day)

100

300

1000

100

300

1000

Body weight

+2

+4

+2

+4

+1

0

- Liver

 

 

 

 

 

 

  . absolute

+6

+5

+10*

+6

+4

+7

  . relative

+3

+1

+8

+3

+3

+6

- Prostate

 

 

 

 

 

 

  . absolute

+4

+13

+9

 

 

 

  . relative

+2

+8

+7

 

 

 

- Testes

 

 

 

 

 

 

  . absolute

-5

-10*

-4

 

 

 

  . relative

-7

-14*

-6

 

 

 

Statistically significant from controls: *: p<0.05.

The significance concerned the organ weights values and not the percentages.

 

Table 3: Number of pups or litters presenting dilated renal pelvis

 

Dose-level (mg/kg)

0

100

300

1000

 

 

 

 

 

# pups evaluated

94

96

76

88

# pups affected (%)(a)

2 (2,1)

4 (4,2)

2 (2,6)

8 (9,1)

 

 

 

 

 

# Litters evaluated

12

12

10

1

# Litters affected (%)(a)

2 (16,7)

3 (25,0)

2 (20,0)

4 (36,4)

 

 

 

 

 

Except where stated: no statistical significance

 

 

(a) Fisher exact test

 

 

 

 

 

Table 4: Historical control values for the distended renal pelvis:

Foetal incidence (%)

 

Litter incidence (%)

AVG

SD

MAX

 

AVG

SD

MAX

1,149

2,06

12,64

 

6,966

11,3

68,18

 
MARTA (1993) Historical Control Data for Development and Reproductive Toxicity Studies using the CrI:CD®BR Rat. Charles River laboratories:  

http://www.criver.com/flex_content_area/documents/rm_rm_r_tox_studies_crlc d_br_rat.pdf).

Applicant's summary and conclusion

Conclusions:
The oral administration of DIMETHYL SULFOXIDE (DMSO) at 100, 300 or 1000 mg/kg/day to male and female Sprague Dawley rats, was well tolerated at all dose-levels. There were no substance-induced effects on the male and female reproductive performance, nor on the progeny of the parental rats up to 1000 mg/kg/day. The no observed effect level (NOEL) for parental toxicity, reproductive performance (mating and fertility) and for toxic effects on the progeny is considered to be 1000 mg/kg/day.
Executive summary:

The potential toxic effects of Dimethylsulfoxide (DMSO) on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition was evaluated in a reproduction/developmental screening study according to the OECD Guideline No. 421 (27th July 1995). Four groups of Sprague Dawley rats (12 males and 12 females) were treated with DMSO at 0, 100, 300, 1000 mg/kg/d as follows: * Males: during 2w before mating, the mating period (2w) and until sacrifice. * Females: during 2w before mating, the mating period (2w), pregnancy (3w), and lactation until day 21 pp inclusive Mortality and clinical signs were checked twice daily. Body weight and food consumption were recorded at regular intervals. Females were paired with males from the same dose-level group until mating occurred. Gestation was monitored. Females were allowed to deliver normally and to rear their progeny until day 21 post-partum. During the lactation period, the pups were examined daily for survival, external abnormalities and clinical signs. At final sacrifice, the pups were examined for gross external abnormalities and then discarded. At final sacrifice of the parent, the testis and epididymides were weighed for the males and a complete macroscopic post-mortem examination was performed for both gender. A microscopic examination was performed on the ovaries, testes and epididymides of females and males, respectively. No death was observed in the parental generation. One female treated at 300 mg/kg/day was sacrificed after the end of the pairing period as no evidence of mating has been observed. No treatment-related clinical signs were observed during the study in males and females. Females of all dose-levels gained slightly less weight (-11%, -26%, -22%) during the pre-pairing period than the controls, although weight gains during gestation and lactation were similar to the controls. No treatment-related effect was observed on mating index, fertility index, duration of gestation and delivery data.
At 100 mg/kg/day, the number of pups dying between days 1 and 4 post-partum was statistically significantly higher than control values. The dead pups were distributed among six litters although as the number of dead pups in the groups treated at 300 or 1000 mg/kg/day was not significantly increased and as the viability index was still 93% and as there were no more deaths after day 4 post-partum, this was considered not to be related to treatment.
Pup necropsy findings were limited to a highest incidence of pups with dilated renal pelvis in the group treated at 1000 mg/kg/day. However, this effect was not statistically significant, in the range of the historical values and there was no dose-relationship between the groups treated at 100 or 300 mg/kg/d.
No effects on mating and fertility or on pre-weaning development of the progeny. The NOAEL for parental toxicity was 1000 mg/kg/day, the NOEL for reproductive performance and for toxic effects on the progeny was 1000 mg/kg/day.