Registration Dossier

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: Guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1991
Reference Type:
publication
Title:
Developmental toxicity of dimethyl sulfate by inhalation in the rat
Author:
Alvarez L, Hurtt ME and Kennedy GL Jr
Year:
1997
Bibliographic source:
Drug|Chem Toxicol 20: 99-114. Cited in: EU RAR (2002)
Reference Type:
publication
Title:
EU Risk Assessment Report Dimethyl Sulphate
Author:
EU RAR
Year:
2002
Bibliographic source:
EU RAR Volume 12. Luxembourg 2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Sulfuric acid, dimethyl ester (synonyms: dimethyl sulphate, DMS, methyl sulfate)
- Analytical purity: 99.9%

Test animals

Species:
rat
Strain:
other: Crl: CD BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Crl:CD BR rats 154 nulliparous ware delivered from Charles River Breeding Laboratories, Ine., Kingston, Nev York, on Kay 14, 1991
- Age at study initiation: females, 63 days old and 82 males, 78 days old
- Weight at study initiation: on the day after delivery, female weights ranged from 179.1 to 246.7 grams with a mean and standard error of 223.6±0.76 grams. Males neighed from 299.4 to 357.4 grams with a mean and standard error of 340.0±1.06 grams
- Housing: individually housed in suspended, stainless steel wire-mesh cages
- Diet (e.g. ad libitum): Purina Certified Rodent Chow 15002 (Meal)
- Water (e.g. ad libitum): water (Wilmington Suburban Water Corporation)
- Acclimation period: 15 days

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

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
nose only
Vehicle:
other: Filtered house line air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: test atmospheres were generated by metering nitrogen over glass impingers containing a reservoir holding DMS.
- Method of holding animals in test chamber: during exposure, 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, thus mininizing any efteets associeted vith prolonged skin contact vith DMS.
- Source and rate of air: filtered, dried dilution air; the dilution air was dried using a glass gas drier containing molecular sieves and dessicated silica in order to prevent the DMS fron hydrolyzing into sulfuric acid and methanol.
- System of generating particulates/aerosols: The nitrogen swept the DNS vapor into each test chamber via teflon tubing. The concentration in each chamber was controlled by varying the flow ot nitrogen through the impinger.
- Temperature, humidity, pressure in air chamber: the impingers were kept in a 25°C water bath and could be isolated from the rest of the generation system using stainless steel plug valves.
- Air flow rate: air was introduced into each chamber at 40 L/min
- Air change rate: the chanbers were operated in a one-pass, flow-through mode with air flow rates adequate to provide 16 air changes per hour. The flow rates provided sufficient oxygen tor the test animals and adequate distribution of DMS in the chambers.
- Treatment of exhaust air: chamber exhaust was bubbled through a 5% ammonia solution and a 1M sodium thiosulfate solution followed by an MSA charcoal and particulate filter before being exhausted into a fume hood. Before transfer to housing facilities at the end of each exposure, test animals were left in their respective chamber's for approximately 10 minutes to allow clearance of test material from the chamber.

TEST ATMOSPHERE
- Brief description of analytical method used: at approximately 60-minute intervals, chamber samples of test atmospheres were bubbled through hexane-tilled impingers by a vacuum pump. Duplicate or triplicate analyses of these samples was carried out using a Hewlett Packard 5880 gas chromatograph equipped with an electron capture detector.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were chromatographed at 65°C on a 15 meter, 0.53 mm O.D., SPB-5 capillary column. The atmospheric concentration of DMS was determined by comparison of the detector response of chamber samples with that of a liquid standard established by injecting a known volume of DMS into hexane.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: females were cohabited with males until copulation was confirmed
- Further matings after two unsuccessful attempts: yes
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy:
Duration of treatment / exposure:
day 6-15 of gestation
Frequency of treatment:
daily; 6 hours/day
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 0.1, 0.7 and 1.5 ppm
Basis:
nominal conc.
target concentration
Remarks:
Doses / Concentrations:
0, 0.12 ±0.05, 0.77 ±0.15 and 1.43 ±0.28 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
25 pregnant animals
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A pilot study was conducted to determine the "maximum tolerated dose" In groups of seven pregnant rats exposed to DMS for six hours daily on Days 7-16 of gestation at nominal concentrations of 0, 0.05, 0.2, 0.5 and 1.0 ppm. Calculated concentrations (± standard deviation) were 0, 0.05±0.038, 0.18±0.113, 0.71±0.803 and 1.2±0.787, respectively. No significant differences were detected in maternal body weight changes except for a significant decrease at the 0.5 ppm level on Days 7-17; however, significant trends were detected on Days 7-9 and 7-17 of gestation. Although no significant differences in feed consumption were seen, significant trends were seen for most of the weighing intervals (Days 7-9, 9-11, 11-13 and 13-15) and for the overall exposure period, Days 7-17. No effects were seen on reproductive or fetal parameters. Based an these data, and considering the small group size and the variability of the data, the exposure levels tabulated above were selected for the study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: observations for morbidity and mortality were made daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical signs were recorded on the day after arrival and three times before mating, and then each morning on Days 1-22 and each afternoon on Days 7-16 (the dosing period).

BODY WEIGHT: Yes
- Time schedule for examinations: body weight was recorded on the day after arrival and three times before mating; females selected for the study were weighed on Days 1, 7, 9, 11, 13, 15, 17 and 22 of gestation.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 22 of gestation by the inhalation of carbon dioxide
- Organs examined:

OTHER:
Sacrifice animals were examined for gross pathologic changes. The gravid uterus was removed and weighed. The uterus was opened and the types of nidations (live and dead fetuses, and resorptions) and their relative positions were recorded. The empty uterine weight was then recorded. The uterus of each apparently “nom-pregnant” rat was opened and stained with ammonium sulfide to detect very early resorptions; data collected were used only to determine the incidence of pregnancy and the number of females with total resorptions. Corpora lutea were counted and the number recorded for each ovary.
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
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
Live fetuses were weighed, sexed and examined for external alterations. For each litter, the maximum stunted weight (MSW) was calculated by subtracting the lightest weight from the total weight, dividing by the remaining number of fetuses and multiplying by 0.666. A fetus weighing the same or less than the MSW was considered stunted; its weight was omitted when the mean litter weight was calculated. If the lightest fetus was determined to be stunted, the procedure was repeated until all remaining fetal weights were in excess of the MSW. The first live fetus and thereafter every other fetus in each litter were decapitated and examined for visceral alteration and the sex verified. Retarded renal development was classified using the schema of Woo and Hoar. The heads were fixed in Bauin's fluid and examined. All stunted and externally malformed fetuses were also examined for visceral alterations; a decision to do a head examination was made on an individual basis.

The remaining fetuses were sacrificed by an intraperitoneal injection of sodium pentobarbital (Sodium Pentobarbital Injection, C-II, Lot No. 8432, Henry Schein, Inc., Port Washington, N.Y.). All fetuses were fixed in 70% ethanol, eviscerated (if not done earlier during visceral examination), macerated in 1% aqueous potassium hydroxide solution, stained with alizarin red S and examined for skeletal alterations.
Statistics:
Incidence of pregnancy, clinical observations, maternal mortality, litters with total resorptions (Test for linear trend: Cochran-Armitage; Pair-wise test between groups: Fisher’s exact), maternal weight, maternal weight change, maternal feed consumption (Test for linear trend: linear combination of dose ranks from ANOVA; Pair-wise test between groups: Dunnett’s when one-way ANOVA was significant); live fetuses, dead fetuses, resorptions, nidations, corpora lutea, fetal weight, incidence of fetal alterations (Test for linear trend: Jonckheere’s; Pair-wise test between groups: Mann-Whitney U). When more than 752% ties occurred in reproductive and fetal parameters, the Cochran-Armitage test replaced Jonckheere's test to detect trend and the Fisher's exact test was applied instead of the Mann-Whitney U test to detect a significant difference between groups. When Bartlett's test for homogeneity of variances was significant analyses of maternal body weights and feed consumption were conducted on the ranks of the original values. The use of the words “significant” or “significantly” in this report indicates a statistically significant difference between the control and the experimental groups.
Indices:
none
Historical control data:
Similar studies are continuously conducted in the test laboratory

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY
All females survived to scheduled sacrifice on Day 22 of gestation.

BODY WEIGHT CHANGES
There was a significant reduction in body weights observed in the high concentration group during Days 9-11, 11-13, 13-15, 15-17 with a resultant reduction on Days 7-17. In the intermediate level significant reductions were detected on Days 13-15 and 7-17. A significant trend accompanied the changes.
At the high level a significant reduction in mean maternal adjusted body weight was seen; this resulted in significant reductions in mean maternal weight gains calculated with the adjusted weight for the periods 1-22 and 7-22. Each of these reductions was accompanied with significant trend.
No other effects on body weights were seen

FEED CONSUMPTION
Feed consumption in the high dose group was reduced during all measurement intervals of the dosing period, resulting in a significant decrease for the overall period. The decreases were significant during Days 9-11, 11-13, 13-15 and 15-17.
At the intermediate level significant decreases were seen on Days 13-15 and 15-17. A significant trend was observed far each measurement interval of the dosing period, as well as for the overall dosing period. No other effects on feed consumption were seen.

CLINICAL OBSERVATIONS
No compound-related effects on the incidence of clinical observations were seen. Other than alopecia, most of the affected animals were found to have signs related to the stress of restraint during the exposures (facial, periocular and perinasal staining).

POSTMORTEM FINDINGS
No compound-related effects were observed during the gross postmortem examinations.

REPRODUCTIVE EFFECTS
With the exception of a slight, but non-significant increase in 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.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
0.77 ppm
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
BODY WEIGHT
Mean fetal body weight at the high exposure level shoved a slight, but non-significant, decrease. No other effects on fetal body weights were seen.

MALFORMATIONS
No effects on the incidence of fetal malformations were seen.

VARIATIONS
No effects on the overall incidence of fetal variations or on the incidence of developmental variations or variations due to retarded development were seen.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1.43 ppm
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

-

Applicant's summary and conclusion