Dimethyl sulphate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data available

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1997

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of pregnant rats were exposed to DMS by inhalation from gestation day 6 to 17 and the fetuses were examined for developmental toxicity.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl sulphate

EC Number:

201-058-1

EC Name:

Dimethyl sulphate

Cas Number:

77-78-1

Molecular formula:

C2H6O4S

IUPAC Name:

dimethyl sulfate

Details on test material:

- Name of test material (as cited in study report): Dimethyl sulphate
- Physical state: clear, oily liquid
- Analytical purity: > 99.5%
- Impurities (identity and concentrations): no specific contaminants were identified
- Stability under test conditions: material was stable throughout the experimental period, as determined by chomatographic analyses
- Storage condition of test material: material was stored in a well-ventilated area away from heat and direct sunlight
- Other: samples was obtained from the Agricultural Products Department, the DuPont Company
No further details are given.

Test animals

Species:

rat

Strain:

other: Crl:CD BD nulliparous

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, NY
- Age at study initiation: 63 days (females), 78 days (males)
- Weight at study initiation: 179-248 g (females), 299-357 g (males)
- Housing: each rat was housed individually in suspended, wire mesh cages of stainless steel.
- Diet: ad libitum, Purina Certified Rodent Chow #5002
- Water: ad libitum
- Acclimation period: animals were quarantined for one week prior to testing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light):12/12

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

nose only

Vehicle:

other: air

Details on exposure:

Groups of 25 pregnant rats were targeted for exposure to DMS. During the exposures, each rat was individually confined in a polycarbonate restrainer placed in a glass and stainless steel, 150-liter chamber so that only the nose of the rat protruded into the chamber. The position of the animals within the chambers were rotated daily to minimise any possible effects due to uneven vapour distribution. The chamber air flow rate was 16 air changes per hour.
Chamber exhaust was bubbled through a 5% ammonia solution and a 1M sodium thiosulphate solution followed by an MSA charcoal and particulate filter before being exhausted into a fume hood.
Test atmospheres were generated by metering nitrogen over glass impingers containing a reservoir holding DMS. Filtered, dried dilution air was introduced into each chamber at 40L/min.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration in each chamber was controlled by varying the flow of nitrogen through the impinger. The impingers were kept in a 25°C water bath.
At approximately 60-minute intervals, chamber samples of test atmosphere were bubbled through hexane-filled impingers by a vacuum pump. Duplicate or triplicate analyses of these samples were carried out using a Hewlett Packard 5880 gas chromatograph. Samples were chromatographed at 65°C on a 15 meter, 0.53 mm O.D. SPB-5 capillary column.
Analytical measurments showed actual concentrations of 0.12 +/- 0.05, 0.77 +/- 0.15 and 1.43 +/- 0.28 ppm.

Details on mating procedure:

The female rats were cohabited overnight with mature males (1/sex/cage). Mating was verified each morning by detection of a copulation plug. Mated females were stratified by body weight and assigned to groups by random sampling within each stratum (25 per group).

Duration of treatment / exposure:

from gestation day 7 to 16

Frequency of treatment:

daily, 10 exposures

Duration of test:

6 hours per day

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 ppm
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.12 +/- 0.05 ppm
Basis:
analytical conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0.77 +/- 0.15 ppm
Basis:
analytical conc.

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1.43 +/- 0.28 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

25 pregnant rats

Control animals:

yes

Details on study design:

No further details.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
Clinical signs were recorded on the day after arrival and three timws before mating. Observations for morbidity and mortality were made daily.

DETAILED CLINICAL OBSERVATIONS: Yes
For females selected for the study, individual clinical signs were recorded each morning throughout the study and each afternoon on day 7-16 (the exposure period).

BODY WEIGHT: Yes
Body weights were recorded on the day after arrival and 3 times before mating. Females selected for the study were weighed on gestation days 1, 7, 9, 11, 13, 15, 17 and 22.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Feed was weighed on days 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 22 of gestation.

WATER CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 22; dams were euthanised with carbon dioxide, and a gross pathologic examination was made.
- Organs examined: uterus

Ovaries and uterine content:

The uterus was opened and the numer and position od all live, dead and resorbed conceptuses were recorded. Both gravid and empty uteri were weighed. The uterus of each apparently non-pregnant dam was stained with ammonium sulphide to determine wether any very early resorption could be identified. The number of corpora lutea were recorded for each ovary.

Fetal examinations:

All fetuses were weiged, sexed and examined for external alterations. The first live fetus and therafter each alternating fetus in each litter were decapitated and examined for visceral alterations and sex verified. Retarded renal development was classified. The remaining fetuses were sacrificed by an intraperitoneal injection of sodium pentobarbital. All fetuses were fixed in 70% ethanol, eviscerated, macerated in 1% aqueous potassium hydroxide solution, stained and examined for skeletal alterations.

Statistics:

The litter was considered the experimental unit for the purpose of statistical evaluation. The level of significance selected was alpha <0.05.
Maternal weight, maternal weight changes and maternal feed consumption: linear contrast of means trend test/Dunnett's when one way ANOVA was significant
Incidence of pregnancy, clinical observations, maternal mortality and litters with total resorptions: Cochran-Armitage/Fisher's exact test
Live fetuses, dead fetuses, resorptions, nidations, corpora lutea, fetal weight and incidence of fetal alterations: Jonckheere's/Mann-Whitney U test

Indices:

no data

Historical control data:

no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: body weight and food consumption

Details on maternal toxic effects:
All female rats survived the testing period. Body weight gain in maternal rats exposed to either 0.7 or 1.5 ppm was reduced, while no effects were seen at 0.1 ppm (Table 1). Feed consumption was reduced in rats of the 0.7 group from day 13 to 17 of exposure and from day 9 through 17 in the 1.5 ppm group. Overall daily mean feed consumption during exposure period was 23.1, 24.3. 21.8 and 18.3 g in the control and 0.1, 0.7 and 1.5 ppm groups, respectively.
A number of animals in each group (3-4) displayed alopecia during the test. The degree of involvement was generally minimal, and there were no essential differences between DMS-treated and control rats. No compound-related effects on the incidence of clinical observations were seen. Most of the affected animals were found to have signs related to the stress of restraint during the exposures (facial, periocular and perinasal staining).

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

0.12 ppm (analytical)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

> 1.43 ppm (analytical)

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 adverse reproductive effects related to DMS exposure were detected (Table 2). With the exception of a slight, but non-significant increase in no. of resorptions at the highest exposure level resulting in a slight decrease in the mean number of live fetuses per litter, no effects on reproductive parameters (pregnancy rate, females with total resorptions, means per litter for live fetuses, resorptions, nidations or corpora lutea) were seen. Fetal weights tended to be slightly reduced at 1.5 ppm, however no statistical significance (p>0.05) was attained. There was no significant difference between the control and experimantal groups in the incidence of external, visceral or skeletal malformations (Table 3). Developmental variations (Table 4) among fetuses derived from DMS-exposed females were not significantly different from those of the controls.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1: Maternal* body weight changes in rats exposed to DMS by inhalation

Period of gestation	Exposure groups
	Control	0.1 ppm	0.7 ppm	1.5 ppm
Days 1-7	42.6 ± 1.8^b	41.2 ± 1.8	39.4 ± 2.5	38.3 ± 2.5
Days 7-9	-4.3 ± 1.0	-3.5 ± 1.9	-5.2 ± 2.4	-5.5 ± 2.0
Days 9-11^d	7.3 ± 1.2	8.5 ± 1.2	5.3 ± 1.6^c	0.4 ± 1.8^c
Days 11-13^d	9.6 ± 1.4	9.7 ± 1.3	9.6 ± 1.3	3.5 ± 1.5^c
Days 13-15^d	9.8 ± 1.8	6.9 ± 1.9	3.7 ± 1.1^c	3.2 ± 1.3^c
Days 15-17^d	18.3 ± 1.3	22.3 ± 2.0	15.7 ± 1.5^c	10.6 ± 2.1^c
Days 7-17^d	40.7 ± 2.0	43.8 ± 3.1	29.2 ± 3.0^c	12.2 ± 3.5^c
Days 17-22	94.6 ± 2.6	91.3 ± 3.8	98.7 ± 4.5	102.7 ± 3.2

* Only data obtained from females having at least one fetus that was alive in utero were included in this table; b: Value presented as means +/- SE; c: Significantly different from controls (Dunnett's test), p<0.05; d: Significant trend (linear combination of dose ranks from ANOVA, p<0.05.

Table 2: Reproduction parameters in dams exposed to DMS by inhalation

	Exposure groups
	Control	0.1 ppm	0.7 ppm	1.5 ppm
Number of pregnant/number treated	^23/25	24/25	25/25	23/25
Corpora lutea/dam	18.0 ± 0.5	18.7 ± 0.6	18.4 ± 0.5	18.2 ± 0.4
Implants/dam	17.0 ± 0.5	16.9 ± 0.8	16.8 ± 0.5	16.7 ± 0.4
Resorption/dam	0.6 ± 0.2	0.7 ± 0.2	0.5 ± 0.1	1.2 ± 0.3
Live fetuses/litter^b	16.4 ± 0.5	16.2 ± 0.8	16.2 ± 0.5	15.6 ± 0.5
Fetal body weight^c(g)	5.35 ± 0.09	5.42 ± 0.11	5.41 ± 0.05	5.24 ± 0.06
No. of stunted fetuses	0	1	3	0

Values are presented as means +/- Se; b: All, with the exception of 1 in the 1.5 ppm group, were classified as early resorptions; c: Stunted fetuses removed from this calculation.

Table 3: Incidence of fetal malformations from dams exposed to DMS by inhalation

Exposure (ppm)	0	0.1	0.7	1.5
External No. examined^a,b No. affected Mean percent affected per litter (SE) Subcutis-anasarca	377 (23) 0 (0) 0.0 ...^d	389 (24) 1 (1) 0.2 (0.22) 1 (1)	406 (25) 0 (0) 0.0 ...	358 (23) 0 (0) 0.0 ...
Visceral No. examined No. affected Mean percent affected per litter	193 (23) 0 (0) 0.0	204 (24) 0 (0) 0.0	210 (25) 0 (0) 0.0	183 (23) 0 (0) 0.0
Head No. examined No. affected Hydrocephaly Encephalocele Mean percent affected per litter (SE)	194 (23) 2 (1) 1 (1) 1 (1) 1 (1.0)	202 (24) 0 (0) ... ... 0.0	210 (25) 0 (0) ... ... 0.0	183 (23) 0 (0) ... ... 0.0
Skeletal No. examined No. affected Mean percent affected per litter	377 (23) 0 (0) 0.0	389 (24) 0 (0) 0.0	406 (25) 0 (0) 0.0	358 (23) 0 (0) 0.0
Total number affected	2 (1)	1 (1)	0 (0)	0 (0)
Mean percent affected per litter )SE)	0.5 (0.54)	0.2 (0.22)	0.0	0.0

a: No significant trend (Cochran-Armitage test) or significant difference from controls (Fisher's exact test); b: expressed as fetuses (litters); c: In calculating the percent affected per litter, dead fetuses are omitted from the numebr examined and the number affected; d: For ease of reading, zeros have been replaced with ... for listed alterations.

Table 4: Incidence of fetal variations from dams exposed to DMS by inhalation

Exposure (ppm)	0	0.1	0.7	1.5
Development variations
External No. examined^a,b No. affected^b Mean percent affected^c per litter	377 (23) 0 (0) 0.0	389 (24) 0 (0) 0.2 (0.22)	406 (25) 0 (0) 0.0	358 (23) 0 (0) 0.0
Visceral No. examined No. affected Mean percent affected per litter (SE) Pulmonary arteries-common trunk	193 (23) 2 (2) 1.0 (0.68) 2 (2)	204 (24) 1 (1) 0.5 (0.46) 1 (1)	210 (25) 0 (0) 0.0 (0.0) ...^d	183 (23) 0 (0) 0.0 (0.0) ...
Head No. examined No. affected Mean percent affected per litter (SE)	194 (23) 0 (0) 0.0	202 (24) 0 (0) 0.0	210 (25) 0 (0) 0.0	183 (23) 0 (0) 0.0
Skeletal No. examined No. affected Mean percent affected per litter (SE) Rib-rudimentary cervical sternum -          Misaligned sternebra -          Fused	377 (23) 2 (2) 0.4 (0.30) 1 (1) ... 1 (1)	389 (24) 0 (0) 0.0 (0.0) ... ... ...	406 (25) 3 (3) 0.7 (0.39) 2 (2) 1 (1) ... 3 (3)	358 (23) 3 (1) 0.8 (0.77) 3 (1) ... ... 3 (1)
Total with developmental variations	4 (4)	1 (1)	3 (3)	3 (1)
Mean percent affected per litter (SE)	1.0 (0.45)	0.2 (0.25)	0.7 (0.39)	0.8 (0.77)
Variations due to retarded development
External No. examined No. affected Mean percent affected per litter	377 (23) 0 (0) 0.0	389 (24) 0 (0) 0.0	406 (25) 0 (0) 0.0	358 (23) 0 (0) 0.0
Visceral No. examined No. affected Mean percent affected per litter (SE) Kidney  - Small papilla, size 1 - Small papilla, size 2	193 (23) 15 (8) 7.8 (2.5) 3 (3) 12 (7)	207 (24) 20 (12) 10.8 (3.11) 6 (5) 14 (10)	210 (25) 9 (9) 4.6 (1.29) 2 (2) 7 (7)	183 (23) 14 (9) 7.4 (2.39) 6 (4) 8 (7)
Head No. examined No. affected Mean percent affected per litter	194 (23) 0 (0) 0.0	202 (24) 0 (0) 0.0	210 (25) 0 (0) 0.0	183 (23) 0 (0) 0.0
Skeletal No. examined No. affected Mean percent affected per litter (SE) Rib – Wavy Skull – Partially ossified Hyoid – Unossified Sternum – Partially ossified - Unfossified Vertebrae – Partially ossified	377 (23) 26 (11) 6.6 (1.83) ... 8 (3) 3 (2) 3 (2) ... 12 (7)	389 (24) 30 (13) 7.5 (2.04) ... 8 (6) ... 5 (2) ... 19 (9)	406 (25) 29 (14) 7.5 (1.82) 2 (1) 9 (6) 5 (3) 2 (2) ... 13 (6)	358 (23) 25 (9) 7.3 (2.65) 1 (1) 5 (4) 1 (1) 2 (1) 1 (1) 17 (8)
Toltal with variations due to retarded development	41 (6)	49 (19)	37 (18)	37 (14)
Mean percent affected per litter (SE)	10.6 (2.17)	13.2 (2.30)	9.7 (2.06)	10.5 (2.90)
Total number with variations	44 (17)	50 (19)	40 (18)	39 (14)
Mean percent of foetuses per litter with variations (SE)	113 (2.75)	13.4 (2.34)	10.4 (2.13)	11.1 (3.14)

a: No significant trend of significant differences from controls; b: Expressed as fetuses (litters); c: In calculating the percent affected per litter, malformed and dead fetuses are omitted from the number examined and the number affected; d: For ease of reading, zeros have been replaced with ... for listed alterations.

Applicant's summary and conclusion

Conclusions:

It can be concluded that DMS is not teratogenic or embryo-/fetotoxic following inhalation exposure of dams during organogenesis at nominal concentrations up to 1.5 ppm.

Executive summary:

In pregnant rats exposed to 0.77 and 1.43 ppm by inhalation, a decrease in food consumption and weight gain of dams was reported. The NOAEL for maternal toxicity was established at 0.12 ppm. No significant differences in malformations and variations were reported between the fetuses in the control and the experimental groups. At the highest concentration tested, a very slight (not statistically significant) decrease of fetal weights was reported which was regarded as not of toxicological relevance. Therefore, it was concluded that the NOAEL can be expected above the highest concentration tested (>1.43 ppm).