Dimethyl disulphide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-06-27 to 2006-09-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl disulphide

EC Number:

210-871-0

EC Name:

Dimethyl disulphide

Cas Number:

624-92-0

Molecular formula:

C2H6S2

IUPAC Name:

(methyldisulfanyl)methane

Details on test material:

- Source: Odor Tech, Pineville, Louisiana,
- Purity test date: 99.8%
- Lot/batch No.: 05.05.05

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina,
- Age : 70 days old upon receipt.
- Weight at study initiation: 218 g to 290 g on gestation day 0.
- Housing: individually housed in clean, stainless steel wire-mesh cages suspended above cage-board.
- Diet : PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002, ad libitum
- Water : Reverse osmosis-purified (on-site) drinking water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS (NON-EXPOSURE PERIODS)
- Temperature : 22°C ± 3°C
- Humidity: 50% ± 20%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

TREATMENT REGIMEN
Exposures were conducted in 2.0-m3 stainless steel and glass whole-body exposure chambers. The chambers were operated under dynamic conditions, at a slight negative pressure with approximately 12-15 air changes per hour.

EXPOSURE ATMOSPHERE GENERATION AND MONITORING
Vapors of the test article were generated using a bubbler type (gas washing bottle) vaporization system. The test article vapors were then directed to the exposure chamber inlet where vapor concentration was reduced to the desired level by mixing with the chamber ventilation air. The control group was exposed to clean, filtered air.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Actual exposure concentrations within each chamber (approximate animal-breathing zones) were measured at least 10 times (approximately every 35 minutes) during each daily exposure period by a validated gas chromatographic method. At least 1 standard was analyzed each day prior to exposure to confirm gas chromatographic calibration.
Actual exposure concentrations within each chamber (approximate animal-breathing zones) were measured at least 10 times (approximately every 35 minutes) during each daily exposure period by a validated gas chromatographic method. At least 1 standard was analyzed each day prior to exposure to confirm gas chromatographic calibration. Overall mean measured exposure concentrations were 0.0, 5.0, 20.2 and 80.0 ppm for the same respective target exposures.

Details on mating procedure:

At the conclusion of the acclimation period, all available females (approximately 12 weeks old) were weighed and examined in detail for physical abnormalities. Each animal judged to be in good health and meeting acceptable body weight requirements (a minimum of 220 g) was placed in a suspended wire-mesh cage with a resident male from the same strain and source for breeding. Resident males were untreated, sexually mature rats utilized exclusively for breeding. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. The day on which evidence of mating was identified was termed gestation day 0.

Duration of treatment / exposure:

gestation days 6-19

Frequency of treatment:

6 hours per day

Duration of test:

sacrifice on GD 20

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 ppm

Doses / concentrations 2

Dose / conc.:

20 ppm

Doses / concentrations 3

Dose / conc.:

80 ppm

No. of animals per sex per dose:

27

Control animals:

yes, sham-exposed

Examinations

Maternal examinations:

CLINICAL OBSERVATIONS AND SURVIVAL: yes
All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual detailed clinical observations were recorded from gestation days 0 through 20 (prior to exposure during the treatment period). Animals were also observed for signs of toxicity at the midpoint of exposure (if visible through the chamber windows) and approximately 1 hour following completion of exposure. All significant findings were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0 and 6-20 (daily).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule : gestation days 0 and 6-20 (daily)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION : no

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Thoracic, abdominal and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined.

Ovaries and uterine content:

LAPAROHYSTERECTOMY
- The number of corpora lutea on each ovary was recorded.
- The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions and the total number of implantation sites were recorded. The individual uterine distribution of implantation sites was documented. The placentae were also examined.

GRAVID UTERINE WEIGHTS
Gravid uterine weight was collected and net body weight (the gestation day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0-20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

Fetal examinations:

- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, all per litter
- Skeletal examinations: Yes, all per litter
- Head examinations: Yes, one-half per litter in Bouin's fixative for subsequent soft-tissue examination by the Wilson sectioning technique. The heads from the remaining one-half of the fetuses were examined by a mid-coronal slice.

Statistics:

Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test article-exposed group to the control group.
Mean maternal body weights (absolute and net), body weight changes (absolute and net) and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test article-exposed groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal and combined) and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test article-exposed groups to the control group.

Historical control data:

YES

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No test article-related clinical findings were noted at the daily examinations, at the midpoint of exposure or 1 hour following the exposure period at any dosage level.

Description (incidence and severity):

n/a

Mortality:

no mortality observed

Description (incidence):

All females in the control, 5, 20 and 80 ppm groups survived to the scheduled necropsy on gestation day 20.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A test article-related mean body weight loss during gestation days 6-9 and lower mean body weight gains during gestation days 9-12, 12-20 and 6-20 (the entire exposure period) were noted in the 80 ppm group compared to the control group; the differences were statistically significant (p<0.01). The decrements in body weight gain throughout the exposure period resulted in mean body weights that were 5.6% to 9.5% lower (p<0.01) than control group values during gestation days 10-20. Also in the 80 ppm group, mean net body weight was 8.5% lower and mean net body weight gain was lower compared to control group values; the differences were statistically significant (p<0.01).
Mean gravid uterine weight at 80 ppm was also statistically significantly (p<0.01) lower than the control group value, corresponding to lower mean fetal weights observed in this group.

In the 20 ppm group, mean body weight gains were slightly lower than the control group during gestation days 6-9 and 9-12; the differences were statistically significant (p<0.05 or p<0.01). Mean body weight gains in the 20 ppm group were similar to control group
values during gestation days 12-20 and when the entire exposure period (gestation days 6-20) was evaluated. The test article-related lower mean body weight gains in this group noted during the first week of exposure were not of sufficient magnitude to result in lower mean body weights. Mean net body weight, net body weight gain and gravid uterine weight in this group were similar to control group values.

Mean maternal body weights, body weight gains, net body weight, net body weight gain and gravid uterine weight in the 5 ppm group were unaffected by test article exposure. Differences from the control group were slight and generally not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 80 ppm group was statistically significantly lower (p<0.01) than control group values during gestation days 6-9, 9-12, 12-20 and when the entire exposure period (gestation days 6-20) was
evaluated. These decrements in food consumption were considered test article-related and corresponded to the mean body weight loss and lower body weight gains noted for this group during the same intervals.

In the 20 ppm group, mean food consumption was similar to the control group during gestation days 6-9, but test article-related, slightly lower (p<0.05 or p<0.01) mean food consumption was noted during gestation days 9-12. This transient decrease in mean food consumption corresponded to the lower mean body weight gain at 20 ppm from gestation days 9-12. Examination of food consumption and body weight data for individual animals during this interval revealed no consistent trends linking low food consumption to reduced body weight gain (or body weight loss) at 20 ppm. Based on the variability of the individual animal data and the similarity of mean food consumption values to control group values during gestation days 12-20 and over the entire exposure period (gestation days 6-20), the slightly lower mean food consumption noted from gestation days 9-12 was not considered adverse at this exposure level.

Food consumption in the 5 ppm group was unaffected by test article exposure. Differences from the control group were slight and generally not statistically significant

Description (incidence and severity):

n/a

Description (incidence and severity):

n/a

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

At the scheduled necropsy on gestation day 20, no test article-related internal findings were observed at exposure levels of 5, 20 and 80 ppm.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

n/a

Description (incidence and severity):

n/a

Effect levels (maternal animals)

Dose descriptor:

NOAEC

Remarks:

Maternal toxicity

Effect level:

20 ppm (analytical)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean fetal weight in the 80 ppm group (3.0 g) was lower than the concurrent control group value (3.7 g) and the minimum mean value in the WIL historical control data (3.4 g). The difference from the concurrent control group was statistically significant (p<0.01) and was considered test article-related.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Description (incidence and severity):

n/a

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were noted for 1, 0, 1 and 2 fetuses in the control, 5, 20 and 80 ppm groups, respectively, and included the following. Microphthalmia (left orbit appeared smaller than normal) in fetus no. 31383-03 in the control group and fetus nos. 31480-13 and 31480-18 in the 80 ppm group. Fetus no. 31480-13 also had anal atresia and vertebral agenesis (all vertebrae posterior to lumbar vertebra no. 4 absent). The only other external malformation observed in this study, localized fetal edema (neck and thorax), was noted for fetus no. 31402-17 in the 20 ppm group. Because these external malformations were observed in single fetuses, were also observed in the control group, and/or were observed in a manner that was not related to maternal exposure concentration, none were considered test article-related.
No external developmental variations were noted for fetuses in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The only fetal skeletal malformation in this study, sternoschisis (sternal band nos. 1-6 not joined), was noted for a single fetus (no. 31444-04) in the 20 ppm group. Because no skeletal malformations were noted at 80 ppm, this malformation was not considered test article-related.
Test article-related differences in the mean litter proportions of skeletal developmental variations were noted in the 80 ppm group. These differences included increased mean litter proportions of unossified sternebrae nos. 5 and/or 6, unossified sternebrae nos. 1, 2, 3 and/or 4, reduced ossification of the vertebral arches, unossified pubis and unossified hyoid, and a decreased mean litter proportion of ossified cervical centrum no. 1 at 80 ppm. Only the difference for unossified sternebrae nos. 5 and/or 6 was statistically significant (p<0.01) compared to the concurrent control group. These skeletal variations were considered test article-related because they corresponded to the reduced mean fetal body weight at 80 ppm, indicative of developmental delay, and were occasionally outside of the WIL historical control data range. The mean litter proportions of the test article-related skeletal developmental variations are summarized in the text table.
The mean litter proportions of malaligned sternebrae in the 5, 20 and 80 ppm groups (1.0%, 1.7% and 1.3% per litter, respectively) were higher than the concurrent control group value (0.3% per litter), but did not exceed the range of mean values in the WIL historical control data (0.0% to 1.7% per litter) and did not occur in a manner that was exposure-related. Therefore, malaligned sternebrae in these groups were not considered test article-related. Skeletal developmental variations noted in the 5 and 20 ppm groups consisted primarily of unossified sternebrae nos. 5 and/or 6, ossified cervical centrum no. 1, and 14th rudimentary ribs. These variations were not considered test article-related because the mean litter proportions in these groups were similar to control group values.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral malformations consisted of hydrocephaly (increased cavitation of both lateral ventricles and the third ventricle) in control group fetus no. 31467-06, and a malpositioned esophagus (located to the right of the trachea) and lobular dysgenesis of the lungs (all right lobes were fused) in fetus no. 31478-03 in the 5 ppm group. Because no soft tissue malformations were noted in the 20 and 80 ppm groups, the soft tissue malformations noted at 5 ppm were not considered test article-related.
Visceral developmental variations noted in the 5 and 20 ppm groups consisted of renal papillae not developed (Woo and Hoar grade 0) and/or distended ureters, and an accessory spleen. These variations were not considered test article-related because there were no visceral developmental variations noted for fetuses in the 80 ppm group.
Renal papillae not fully developed (Woo and Hoar grade 1) were observed in 1 fetus each in the control, 5 and 20 ppm groups (fetus nos. 31395-05, 31391-06 and 31517-06, respectively). Atrial cysts and a white area on the right atrium were noted for fetus no. 31453-11 in the control group. These findings were not classified as either malformations or developmental variations and were not included in any tabulation.

Details on embryotoxic / teratogenic effects:

The numbers of fetuses (litters) available for morphological evaluation were 405(27), 405(26), 406(27) and 408(26) in the control, 5, 20 and 80 ppm groups, respectively. Malformations were observed in 2(2), 1(1), 2(2) and 2(1) fetuses (litters) in these same respective exposure groups.

Effect levels (fetuses)

Dose descriptor:

NOAEC

Effect level:

>= 80 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on lower mean maternal body weight gains and food consumption noted at 80 ppm, a dosage level of 20 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on the lower mean fetal weight and increased mean litter proportions of several skeletal variations noted at 80 ppm, an exposure level of 20 ppm was also considered to be the NOAEL for prenatal developmental toxicity when pregnant Crl:CD(SD) rats were exposed to dimethyl disulfide via whole-body inhalation exposure for 6 hours daily during gestation days 6-19.

Executive summary:

ln a developmental toxicity study performed following the OECD guideline # 414, four groups of 27 bred female Crl:CD(SD) rats were exposed to either filtered or vapor atmospheres of DMDS for 6 hours daily in whole-body inhalation chambers during gestation days 6 through 19. Test concentrations were 0, 5, 20 and 80 ppm. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each female. The uterus, placenta and ovaries were examined and the number of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were culculated. The fetuses weree weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. The maternal LOAEL was 80 ppm based on lower mean maternal body weight gains and food consumption. The NOAEL for maternal toxicity was 20 ppm. The feta/developmental toxicity LOAEL was also 80 ppm based on lower mean fetal weight and increased mean litter proportions of several skeletal variations. The NOAEL for fetal/developmental toxicity was 20 ppm, no teratogenic effect was observed.