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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

The adverse effects of DMDS on sexual function and fertility were assessed based on results of the two-generation study on rats exposed by inhalation to DMDS vapour performed according to OECD TG 416 (Nemec, 2006b) and on results of the reproduction/developmental toxicity screening inhalation study of DMDS in rats according to OECD TG 421 (Nemec MD, 2006c).


No adverse effects on sexual function and fertility at any exposure concentrations were observed in these both studies.


 

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
2001
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina,
- Age at reception: 31 days old (F0)
- Age at the initiation of test article exposure: 6.6 weeks
- Body weight at exposure initiation: (F0) Males: 211-263 g; Females: 137-178 g
- Housing (non exposure periods): individually in wire-mesh cages suspended above cage-board. After mating and until weaning, the females were transferred to plastic maternity cages with nesting material.
- Diet : PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002, ad libitum. Food was withheld during the exposure periods.
- Water: municipal water, ad libitum. Water was withheld during the exposure periods.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS (NON-EXPOSURE PERIODS)
- Temperature (°C): 22 ± 3 (actual : 21.3°C to 21.5°C)
- Humidity (%): 50 ± 20 (actual: 35.7% to 62.1%)
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 2005-06-28 to 2006-06-09
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
Exposures were conducted in 2.0-m3 stainless steel and glass whole-body exposure chambers. One chamber was dedicated to each group for the duration of the study. The exposure period was 6 hours per day, 7 days per week and was defined as the time between turning the generation system on and off, including the start equilibration time. The chambers were operated under dynamic conditions, at a slight negative pressure (ca 0.5 in of water) with at least 12 to 15 air changes per hour.
Vapors of DMDS were generated using a bubbler-type (gas washing bottle) vaporization system. DMDS 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.
Details on mating procedure:
The animals were paired on a 1:1 basis within each treatment group after a minimum of 70 days of exposure. All animals were randomly selected for pairing, avoiding sibling matings. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. The animals were separated, and the female was housed in an individual plastic cage with nesting material. When evidence of mating was not apparent after 14 days, the female was placed in a plastic maternity cage with nesting material, with no further opportunity for mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Actual exposure concentrations within each chamber were measured at least 10 times (approximately every 35 minutes) during each daily exposure period by a gas chromatographic method. At least 1 standard was analyzed each day prior to exposure to confirm gas chromatographic calibration. Overall mean measured test article exposure concentrations for the F0 generation were 0, 5.0, 20.4 and 79.9 ppm and for the F1 generation were 0, 5.0, 20.6 and 80.0 ppm for the filtered air, 5, 20 and 80 ppm groups, respectively.
Duration of treatment / exposure:
The F0 and F1 males and females were exposed to the test atmosphere for a minimum of 70 consecutive days prior to mating. Exposure of the F0 and F1 males continued throughout mating and through the day prior to euthanasia. The F0 and F1 females continued to be exposed throughout mating and gestation through gestation day 20. To prevent confounding effects on nursing, exposure for F0 and F1 females was suspended from gestation day 21 through lactation day 4, inclusively, and was re-initiated on lactation day 5 and continued through the day prior to euthanasia. During lactation (except when indicated above), the dams were removed from their litters during each daily 6-hour exposure period.
Frequency of treatment:
6 hours per day, 7 days per week
Details on study schedule:
Each generation was mated once to produce 1 litter per generation (the F1 and F2 litters).
Prior to the F0 pairing (week 10), male body weights ranged from 355 g to 623 g and female body weights ranged from 230 g to 363 g. The animals were approximately 17 weeks old. All animals were randomly selected for pairing.
Each F0 dam and litter remained housed together until weaning on lactation day 28. Thirty male and 30 female F1 pups from each group (control, 5, 20 and 80 ppm) were randomly selected prior to weaning (PND 28) to comprise the F1 generation. These pups (a minimum of 1 male and 1 female per litter, when available) were exposed to the test article for 6 hours per day, 7 days per week beginning on PND 28.
Prior to the F1 pairing (week 29), male body weights ranged from 300 g to 631 g and female body weights ranged from 203 g to 364 g. The animals were approximately 15 to 17 weeks old.
Remarks:
Doses / Concentrations:
5, 20 and 80 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
30
Control animals:
yes, sham-exposed
Positive control:
Not appropriate
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity. In addition, the animals were observed for appearance, behavior and pharmacotoxic signs at the midpoint of exposure for animals visible through the chamber windows and 1 hour after completion of exposure for all animals. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed physical examinations were recorded weekly for all parental animals throughout the study period.

BODY WEIGHT: Yes
Individual F0 and F1 male body weights were recorded weekly throughout the study and prior to the scheduled necropsy. Individual F0 and F1 female body weights were recorded weekly until evidence of copulation was observed. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 1, 4, 7, 14, 21 and 28. After weaning (lactation day 28), weekly body weights were recorded for these females until the scheduled necropsy.

FOOD CONSUMPTION: yes
Individual F0 and F1 male and female food consumption was measured weekly until pairing. Food intake was not recorded during the mating period. Following mating, male food consumption was measured on a weekly basis until the scheduled necropsy. Female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17 and 20 and lactation days 1, 4, 7, 14, 21 and 28.

WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily and the slides were evaluated to assess the regularity and duration of the estrous cycles of each F0 and F1 female for 21 days prior to pairing and continuing until evidence of mating was observed or until the end of the mating period.
Sperm parameters (parental animals):
Spermatogenic endpoints (sperm motility including progressive motility, morphology and numbers) were recorded for all surviving F0 and F1 males.
Litter observations:
F0 AND F1 PARTURITION
All females were allowed to deliver naturally and rear their young to weaning (PND 28). During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. Beginning on the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.

STANDARDISATION OF LITTERS
8 pups per litter, 4 per sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters
with fewer than 8 pups. All selections were performed by computerized randomization. The remaining offspring were weighed, euthanized by intraperitoneal injection of sodium pentobarbital and discarded on PND 4.

PARAMETERS EXAMINED IN F1 AND F2 LITTERS
- LITTER VIABILITY AND DEATHS
Each litter was examined twice daily for survival, and all deaths were recorded.
- CLINICAL OBSERVATIONS
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1, 4, 7, 14, 21 and 28. Any abnormalities in nursing behavior were recorded.
- BODY WEIGHTS
Pups were individually weighed on PND 1, 4, 7, 14, 21 and 28.
- SEX DETERMINATION
Pups were individually sexed on PND 0, 4, 21 and 28.
- BALANOPREPUTIAL SEPARATION
Each male pup was observed for balanopreputial separation beginning on PND 35. Examination of the pups continued daily until balanopreputial separation was present. Body weights were recorded at the age of attainment of this landmark.
- VAGINAL PATENCY
Each female pup was observed for vaginal perforation beginning on PND 25. Examination of the females was continued daily until vaginal patency was present. Body weights were recorded at the age of attainment of this landmark.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
All F0 adults were euthanized following the selection of the F1 generation and completion of a detailed clinical observation. All surviving F1 adults were euthanized following weaning of the F2 pups.

GROSS NECROPSY
A complete necropsy was conducted on all parental animals (F0 and F1) found dead, euthanized in extremis or at termination. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities, including viscera.
For females that delivered or had macroscopic evidence of implantation, the numbers of former implantation sites (the attachment site of the placenta to the uterus) were recorded.

ORGAN WEIGHTS
The following organs were weighed from all F0 and F1 parental animals at the scheduled
necropsies: Adrenal glands, Brain, Epididymidesa (total and cauda), Kidneys, Liver, Ovaries, Pituitary gland, Prostate gland, Seminal vesicles with coagulating glands (with accessory fluids), Spleen, Testes(a, Thymus gland, Uterus with oviducts and cervix.
(a = These paired organs were weighed separately.

HISTOPATHOLOGY
Microscopic evaluations were performed on the following tissues for F0 and F1 parental animals from the control and high exposure groups and for all adult animals found dead or euthanized in extremis: Adrenal glands, Brain, Cervix, Coagulating gland, Epididymisa (right): caput, corpus and cauda, Kidneys, Liver, ovaries, Oviducts, Pituitary gland, Prostate gland, Seminal vesicles, Spleen, Testis (right), Thymus gland, Uterus, Vagina, Vas deferens, All gross (internal) lesions (all groups).
Because the adrenal cortex was identified as a potential target tissue, all of the F0 male rats in the 5 and 20 ppm groups were examined in addition to the initial examination of the control and 80 ppm groups.
Postmortem examinations (offspring):
SACRIFICE AND GROSS NECROPSY
All remaining nonselected F1 and F2 weanlings were euthanized and necropsied on PND 28, with emphasis on developmental and reproductive system morphology. All gross lesions from F1 and F2 weanlings and the brain, spleen and thymus from 1 randomly selected weanling per sex per litter were preserved in 10% neutral-buffered formalin for possible future histopathologic examination; all other tissues were discarded. The carcasses were then discarded.

ORGAN WEIGHTS
The following organs were weighed from 1 randomly selected pup per sex per litter for the nonselected F1 and F2 pups: Brain, Thymus, Spleen.
Statistics:
Where applicable, the litter was used as the Parental mating and fertility indices were analyzed using the Chi-square test with Yates’ correction factor. Mean parental (weekly, gestation and lactation) body weights and body weight changes, parental food consumption and food efficiency data, estrous cycle lengths, pre-coital intervals, gestation lengths, implantation sites, live litter sizes, unaccounted sites, numbers of pups born, balanopreputial separation data (day of attainment and body weight), vaginal patency data (day of attainment and body weight), absolute and relative organ weights, sperm production rates, epididymal and testicular sperm numbers, and ovarian primordial follicle counts, 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 was used to compare the test article-exposed groups to the control group. Mean offspring body weights and body weight gains during PND 1-4 and 4-28 were analyzed separately by sex using an analysis of covariance (ANCOVA), with litter size as the covariate. Mean litter proportions (percent per litter) of postnatal pup survival and pup sexes at birth (percentage of males per litter), percentages of motile sperm, and percentages of sperm with normal morphology 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 was used to compare the test article-exposed groups to the control group. Histopathological findings in the test article-exposed groups were compared to the control group using a two-tailed Fisher’s Exact test.
Reproductive indices:
Mating, fertility, copulation and conception indices were calculated.
Offspring viability indices:
Mean Live Litter Size, postnatal Survival Between Birth and PND 0 or PND 4 (Pre-Selection), postnatal Survival for All Other Intervals indices were calculated.
Clinical signs:
no effects observed
Description (incidence and severity):
All F0 parental animals in the control, 5, 20 and 80 ppm groups survived to the scheduled necropsy. No exposure-related clinical findings were noted during the F0 generation at the weekly examinations or at the midpoint exposure and 1 hour following exposure observations.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
* WEEKLY
Reduced (p<0.01) mean body weight gains were noted in the 20 and 80 ppm F0 male groups during study weeks 0-1 (31 g and 14 g, respectively) and 1-2 (40 g and 34 g, respectively) of test article exposure when compared to the control group values of 40 g and 47 g, respectively. Lower mean body weight gain in the 80 ppm F0 male group continued throughout the remaining pre- and post-mating exposure periods when compared to the control group; the differences from the control group values were statistically significant (p<0.05 or p<0.01) during study weeks 2-3, 7-8, 8-9, 9-10, 15-16 and 16-17. Mean body weight gain in the 20 ppm F0 male group was similar to the control group during the remaining pre- and post-mating exposure periods. As a result, cumulative mean body weight gains in the 80 ppm F0 male group were statistically significantly (p<0.01) reduced at 200 g and 258 g during the entire pre-mating (study weeks 0-10) exposure period and during the entire generation (study weeks 0-19), respectively, when compared to a mean body weight gains of 266 g and of 339 g, respectively, in the control group. The cumulative mean body weight changes in the 20 ppm F0 male group during these same exposure intervals were only slightly lower (not statistically significant) when compared to the control group and indicated partial recovery of body weight gain effects noted during the first 2 weeks of exposure.
During the first week of exposure (study week 0-1), mean body weight gains in the 20 and 80 ppm group F0 females (17 g and 12 g, respectively; statistically significant, p<0.05 or p<0.01) were lower when compared to a mean body weight gain of 21 g in the control group. Statistically significant (p<0.05 or p<0.01) reduced mean body weight gains in the 80 ppm F0 female group continued during study weeks 1-2 and 2-3 when compared to the control group (18 g compared to 25 g and 24 g compared to 29 g, respectively). Mean body weight gains in the 20 ppm group were similar to the control group from study weeks 1-2 and statistically significantly lower (p<0.05) during study weeks 2-3 (24 g compared to 29 g). These findings were attributed to the onset of test article exposure. Following the initial 3 weeks of exposure, mean body weight changes in the 20 and 80 ppm group females were similar to the control group during study weeks 3-10. The cumulative mean body weight gains in these respective groups were 144 g and 133 g, respectively, during the pre-mating period (study weeks 0-10) compared to 154 g in the control group. The differences from the control group were statistically significant (p<0.01) in the 80 ppm group and not statistically significant in the 20 ppm group.
Accordingly, reductions in mean body weights (3.6% to 5.3% and 9.9% to 12.8%, respectively; p<0.05 or p<0.01) were observed in the 20 and 80 ppm F0 male groups during the first 3 weeks of exposure when compared to the control group. Statistically significantly lower (11.7% to 15.2%; p<0.01) mean body weights in the 80 ppm F0 male group continued from study week 4 throughout the remaining pre- and post-mating periods when compared to the control group. Mean body weights were only slightly reduced (3.2% to 4.4%; not statistically significant) in the 20 ppm F0 male group during this same interim compared to the control group. When the F0 females were evaluated at 80 ppm, reductions in mean body weights (6.1% to 9.5%; p<0.01) were observed during the entire treatment period when compared to the control group. In addition, mean body weights in the 20 ppm F0 female group were only slightly reduced (3.0% to 5.2%) during the exposure period compared to the control group; differences were statistically significant during study weeks 1 and 3 only. The reductions in mean body weights in the 80 ppm group, and to a lesser extent in the 20 ppm group, were due to exposure-related decrements in the rates of weight gains noted in the first 1 to 3 weeks of the study.
No exposure-related effects on mean body weights or mean body weight gains were observed in the 5 ppm group males and females. Slightly lower mean body weight gains (p<0.05) were noted in these males during study weeks 1-2 and 16-17 when compared to the control group. These differences were not of sufficient magnitude to cause reductions in cumulative mean body weight gains from study weeks 0-10 and 0-19, and therefore, indicated no adverse effect at the lowest exposure concentration.
* GESTATION
No exposure-related effects in mean body weight change were noted in any exposure concentration during the F0 gestation period. Lower mean body weights (6.3% to 7.4%) in the 80 ppm group were observed during the entire gestation period when compared to the control group. The differences from the control group values were statistically significant (p<0.01) and were due to the lower mean body weight gain observed during the pre-mating period. Mean body weights in the 5 and 20 ppm groups were similar to the control values during this exposure period.
* LACTATION
Slightly lower mean body weights (4.8% to 7.4%) in the 80 ppm F0 female group were noted during lactation days 1-28 compared to the mean body weights in the control group; the differences from control values were statistically significant (p<0.05 or p<0.01). These decreased mean body weights were attributed to decreased mean body weight gain during the pre-mating period. No exposure-related effects were noted in mean body weights in the 5 and 20 ppm groups or in mean body weight changes in 5, 20 and 80 ppm during the lactation period when compared to the control group values.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
* WEEKLY
Mean food consumption, evaluated as g/animal/day, was statistically significantly lower (p<0.05 or p<0.01) in the 20 and 80 ppm F0 male groups during study weeks 0-2 when compared to the control group. Mean food consumption, evaluated as g/kg/day, was also statistically significantly lower (p<0.01) in the 80 ppm male group during study weeks 0-1 and in the 20 and 80 ppm male groups (p<0.05 or p<0.01) during study weeks 1-2. These differences were related to the onset of exposure to the test article. Following the first 2 weeks of exposure, mean food consumption in the 20 ppm group males was unaffected by test article exposure, while statistically significantly (p<0.05 or p<0.01) lower mean food consumption, evaluated as g/animal/day, in the 80 ppm F0 males continued throughout the remaining exposure period. However, mean food consumption, evaluated as g/kg/day, in this same group were statistically significantly higher (p<0.01 or p<0.05) during study weeks 3-4, 4-5, 5-6, 6-7, 7-8, 12-13, 13-14, 14-15, 17-18 and 18-19 when compared to the control group. These differences correlated with the lower mean body weights observed in this same group during this exposure interim and were attributed to test article exposure.
Exposure-related effects on mean food consumption were noted to a lesser extent in the 20 and 80 ppm F0 females than the corresponding F0 male groups. Mean food consumption in the 20 ppm (g/animal/day) and 80 ppm (g/animal/day and g/kg/day), group F0 females were statistically significantly lower (p<0.01) during study week 0-1 when compared to the control group. Mean food consumption (g/animal/day) in the 80 ppm group continued to be lower (p<0.01) during study weeks 1-2 and 2-3. These differences were also related to the onset of exposure to the test article and correlated with the reduced mean body weight gain observed in these females during the first 2 weeks of exposure. Mean food consumption (g/animal/day) in the 20 and 80 ppm female groups was similar to the control group values during the remainder of the pre-mating exposure period (study weeks 1-10 for the 20 ppm group females and study weeks 3-10 for the 80 ppm group females). Mean food consumption values (measured as g/kg/day), in these females however, were also higher during this same exposure period when compared to the control group values. The differences were often statistically significant (p<0.05 or p<0.01) and correlated with the lower mean body weights observed in these groups during study weeks 3-10.
Correspondingly, food efficiency was reduced (p<0.01) in the 20 and 80 ppm males and in the 80 ppm females during study weeks 0-1 and in the 80 ppm males and females during study weeks 1-2. There were no other exposure-related effects on food efficiency. No exposure-related effects in mean food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency were noted in the 5 ppm F0 male and female groups when compared to the control group. Slight differences were noted from control values but were not statistically significant and not considered related to test article exposure.
* GESTATION
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency in the 5, 20 and 80 ppm group F0 females were unaffected by test article exposure during gestation. The only statistically significant (p<0.05 or p<0.01) differences from the control group were increased mean food consumption, evaluated as g/kg/day, in the 20 and 80 ppm groups during gestation days 14-17 and decreased mean food consumption, evaluated as g/animal/day, in the 80 ppm group during gestation days 7-11. These slight increases and decreases were considered insignificant or were correlated to slightly lower mean body weights observed in these females during this period.
* LACTATION
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency in the 5, 20 and 80 ppm group F0 females were unaffected by test article exposure during lactation. Differences from the control group were slight and not statistically or biologically significant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Although cytoplasmic vacuolization was noted in the adrenal cortex of males from all exposure levels, including the controls, there was an increased incidence of adrenocortical cytoplasmic vacuolization in males from the 80 ppm group. Histopathologic examination of the adrenal gland of males from the 5 and 20 ppm groups revealed no alteration in the incidence of cytoplasmic vacuolization in the adrenal cortex. The increased level of cytoplasmic vacuolization of the adrenal cortex may be related to the altered adrenal gland organ weights, as discussed above. However, the microscopic changes in the adrenal cortex of the 80 ppm males were not observed in the F1 generation males. Therefore, a definitive resolution of these issues is beyond the scope of the present study.
Other microscopic changes were considered to be incidental findings, manifestations of spontaneous diseases, or related to some aspect of experimental manipulation other than exposure to the test article. There was no exposure-related alteration in the incidence, severity or histologic character of those incidental and spontaneous tissue alterations.
Evaluation of ovarian primordial follicles (10 sections from 10 randomly selected females were evaluated) showed no evidence of an effect on the amount of primordial follicles. The mean number of ovarian primordial follicle counts in the F0 80 ppm group (61.9) was comparable to the control group value (60.9).
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The mean lengths of estrous cycles in the 5, 20 and 80 ppm group were also similar to the control group value. None of these differences were statistically significant.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No exposure-related effects were observed in F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility and morphology) in males at any dosage concentration. Differences from the control group were slight and were not statistically significant.
Reproductive performance:
no effects observed
Description (incidence and severity):
No exposure-related effects on F0 reproductive performance were observed at any concentration. Male and female mating indices were 100.0%, 100.0%, 100.0% and 93.3% in the control, 5, 20 and 80 ppm groups, respectively. Male and female fertility indices were 96.7%, 93.3%, 96.7% and 90.0% and male copulation and female conception indices were 96.7%, 93.3%, 96.7% and 96.4% for the same respective groups.
No statistically significant differences were noted between the control and test article-exposed groups. Males that did not sire a litter numbered 1, 2, 1 and 3 in the control, 5, 20 and 80 ppm groups, respectively. Females that had evidence of mating but did not deliver numbered 1, 2, 1 and 1 in the same respective groups.
The mean numbers of days between pairing and coitus in the test article-exposed groups were similar to the control group value. No test article-related effects on F0 mean gestation length were observed at any exposure level. Mean gestation lengths in the control, 5, 20, and 80 ppm groups were 22.0, 21.8, 21.9, and 21.9 days, respectively.
Key result
Dose descriptor:
NOAEC
Remarks:
reproductive and developmental toxicity
Effect level:
>= 80 ppm (analytical)
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEC
Remarks:
parental systemic toxicity
Effect level:
5 ppm (analytical)
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
No test article-related deaths occurred in the F1 generation.
No exposure-related clinical findings were noted during the F1 generation at the weekly examinations or at the midpoint exposure and 1-hour following exposure observations.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
* WEEKLY
Exposure-related decreased mean body weight gains (statistically significant, p<0.01) were noted in the 5, 20 and 80 ppm male groups and in the 20 and 80 ppm female groups during study week 18-19 and in the 5, 20 and 80 ppm males during study week 19-20. Decreased weekly mean body weight gains in the 20 and 80 ppm males continued throughout the remaining pre-mating and mating periods when compared to the control group; differences from the control were occasionally statistically significant (p<0.05 or p<0.01). Mean body weight gains in the 20 and 80 ppm female groups during this same interval were similar to the control group. As a result of lower gains during study week 18-19, 19-20 and/or the remaining pre-mating period, cumulative mean body weight changes in the 20 and 80 ppm male and 80 ppm female groups were statistically significantly (p<0.01) reduced during the pre-mating period (study weeks 18-30) when compared to the control group. Mean body weight gain in the 5 ppm male group was similar to the control group from study week 20 throughout the remaining treatment period. Cumulative mean body weight gains in this group were similar to the control group values during the pre-mating period (study weeks 18-30) and during the entire generation (study weeks 18-39). The slight, transient differences in male body weight gain from control values at 5 ppm were not considered biologically significant. Mean body weight gain in the 5 ppm female group was unaffected by test article exposure during the entire pre-mating and mating periods.
Mean body weights in the 5, 20 and 80 ppm male groups were statistically significantly (p<0.05 or p<0.01; 19%, 14% and 17%, respectively, at study week 18) lower starting at the onset of exposure and continuing throughout the entire exposure period. Mean body weights in the 5, 20 and 80 ppm females were statistically significantly (p<0.05 or p<0.01; 13.5%, 12.4% and 15.7% at study week 18) lower at the onset of exposure when compared to the control group. Statistically significantly (p<0.01) lower mean body weights in the 80 ppm females continued to be observed throughout the remaining pre-mating and post-mating exposure periods when compared to the control group. During this same interim, lower mean body weights were noted (occasionally statistically significant, p<0.05 or p<0.01) in the 20 ppm females and mean body weights in the 5 ppm females were similar when compared to the control group. The decrements in mean body weights were attributed to test article exposure.
* GESTATION
Mean F1 maternal body weight gains in the 80 ppm group were statistically significantly decreased (p<0.05) when the entire gestation period (days 0-20) was evaluated as a result of lower (p<0.01) mean body weight gain during gestation days 17-20 compared to the control group. Mean body weights in this same group were 13.2% to 15.1% lower than the control group values throughout gestation. The differences from the control group values were statistically significant (p<0.01). These mean body weight decreases noted during gestation were a continuation of the decreases observed in this group during the pre-mating period.
Mean maternal body weights, body weight gains and cumulative body weight changes in the 5 and 20 ppm groups were unaffected by test article administration. Increased (statistically significant, p<0.05) mean body weight gains in the 5 and 20 ppm groups were noted during gestation days 14-17. The increases did not occur in an exposure-related manner and were not considered test article-related.
* LACTATION
A statistically significantly (p<0.01) increased mean body weight gain was observed in the 80 ppm group during lactation days 1-4 when compared to the control group and was considered related to cessation of inhalation exposure during this time. The increase during lactation days 1-4 caused the overall lactation period (lactation days 1-28) to be increased (statistically significant, p<0.01) when compared to the control group. Statistically significantly (p<0.01) lower mean body weight was also noted in the 80 ppm F1 female group during the entire lactation period when compared to the control group. This difference from the control group was attributed to the lower mean body weight gains during the pre-mating period.
Mean body weights and mean body weight gains in the 5 and 20 ppm groups were unaffected by test article exposure. Differences from the control group were slight and not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
* WEEKLY
Mean food consumption, evaluated as g/animal/day, was statistically significantly (p<0.05 or p<0.01) reduced in the 5, 20 and 80 ppm males and in the 20 and 80 ppm females starting during study weeks 18-19 and 19-20. This trend continued throughout the remaining pre-mating period (study weeks 20-30) in the 20 and 80 ppm males and in the 80 ppm females. The differences from control were generally statistically significant (p<0.05 or p<0.01). Food consumption, evaluated as g/kg/day, in 5, 20 and 80 ppm male groups was increased starting at study weeks 20-21 and continuing throughout the entire pre-mating period when compared to the control group. The majority of the differences from control was statistically significant (p<0.05 or p<0.01). Food consumption, evaluated as g/kg/day, in the 20 and 80 ppm female groups was slightly increased (occasionally statistically significant, p<0.05 or p<0.01) starting on study weeks 20-21 and continuing throughout most of the pre-mating period when compared to the control.
Decrements in mean body weights in these animals correlated with these changes in food consumption and indicated that the males appeared to be more sensitive to exposure-related effects than the females.
Statistically significantly (p<0.05 or p<0.01) increased food consumption (g/kg/day) was observed in the 5 ppm females starting on study weeks 18 to 26 and continuing (not statistically significant) throughout the remaining pre-mating period. This finding suggested a compensatory effect that correlated with slightly decreased mean body weight gain and slightly lower mean body weight noted at the initiation of direct exposure followed by normal mean body weights by the third week of exposure. Also correlating to lower mean body weights at the onset of exposure, food efficiency in
the 80 ppm males and females was statistically significantly (p<0.05) lower during study weeks 18-19 for males and females when compared to the control group. Following the initiation of exposure, food efficiency in all test article-exposed groups was generally similar or only slightly lower throughout the remaining pre-mating exposure when compared to the control group.
* GESTATION
Food consumption, evaluated as g/animal/day, was statistically significantly reduced in the 80 ppm group during gestation days 4-20 when compared to the control group. A corresponding statistically significant decrease in mean body weight gain was noted in this group during gestation days 0-20. Food consumption, evaluated as g/kg/day, and food efficiency in the 5, 20 and 80 ppm groups were unaffected by test article exposure.
* LACTATION
Food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency in the 20 and 80 ppm groups were increased during lactation days 1-4; the differences were often statistically significant (p<0.05 or p<0.01) and were attributed to a compensatory effect due to the cessation of test article exposure. Food consumption (g/kg/day) and food efficiency in the 80 ppm group were also increased (p<0.05 or p<0.01) during evaluation of the entire lactation period, days 1-28, when compared to the control group. These findings corresponded with the compensatory higher body weight gain noted in the 80 ppm group during the cessation of test article exposure from lactation days 1-4 and the overall reduced mean body weights in the 20 and 80 ppm animals noted during pre-mating. No differences in food consumption in the 5 ppm group were noted when compared to the control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
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:
effects observed, treatment-related
Description (incidence and severity):
The organ weight changes listed in TABLE 2 were statistically significant when compared to the control group but were generally considered to be a result of exposure-related effect on final body weights. While the lower body weight achieved a level of statistical significance only in males from the 20 and 80 ppm exposure levels and females from the 80 ppm exposure level, final body weight of males from the 5 ppm and females from the 20 ppm groups were also lower than control values. The lower final body weight of 5 ppm males and 20 ppm females influenced the analysis of a number of organ weights, resulting in statistically significant alterations in some organ weights when expressed relative to final body weight.
The organ weight data in the F1 animals indicate, as expected, that adrenal gland and brain weights did not decline in synchrony with body weights. Brain weights of test article-exposed animals were only slightly lower than control values, but the slight difference achieved statistical significance for 5 and 80 ppm males and 80 ppm females. Absolute adrenal gland weights of test article-exposed males remained relatively constant. Adrenal gland weights of test article-exposed females remained relatively constant or, in the case of 80 ppm females, were higher than control values. The final result was that adrenal gland weights of males and females exposed at the 80 ppm level were significantly higher than control values when expressed relative to final body weight or brain weight. This apparent adrenal weight alteration was attributed either directly or indirectly to the test article-related effect on final body weight, with an unknown contribution due to generalized stress and a stress response.
There were no other exposure-related effects on organ weights. A difference in absolute left testis weight at 5 ppm was statistically significant when compared to the control group, but the absolute weight and weights relative to body and brain weight were discordant and were not observed in an exposure-related manner; thus the changes were considered to be spurious.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no exposure-related macroscopic changes noted at the scheduled necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no exposure-related microscopic changes.
A mammary adenocarcinoma was identified in 1 of 30 females from the F1 generation 20 ppm group. Although such a finding is unusual in an animal of that age, it was considered incidental based on its single occurrence and the lack of an exposure-response relationship.
Evaluation of ovarian primordial follicle counts (10 sections from 10 randomly selected females were evaluated) showed no evidence of an effect on the amount of primordial follicles. The mean number of ovarian primordial follicle counts in the F1 80 ppm group (77.9) was comparable to the control group value (61.5).
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The mean lengths of estrous cycles in the 5, 20 and 80 ppm groups were also similar to the control group value. None of these differences were statistically significant.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No exposure-related effects were observed on F1 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility and morphology) in males at any dosage concentration.
Description (incidence and severity):
No exposure-related effects on F1 reproductive performance were observed at any concentration. Male and female mating indices were 93.3%, 89.7%, 100.0% and 93.3% in the control, 5, 20 and 80 ppm groups, respectively. Male and female fertility indices were 83.3%, 86.2%, 93.1% and 90.0%, and male copulation and female conception indices were 89.3%, 96.2%, 93.1% and 96.4% for the same respective groups. No statistically significant differences were noted between the control and test article-exposed groups. Males that did not sire a litter numbered 5, 4, 2 and 3 in the control, 5, 20 and 80 ppm groups, respectively. The numbers of females that had evidence of mating but did not deliver were 3, 1, 2 and 1 in the same respective groups.
The mean numbers of days between pairing and coitus in the test article-exposed groups were similar to the control group value.
No exposure-related effects were noted on mean gestation lengths or the process of parturition at any concentration. Mean F1 gestation lengths in the control, 5, 20 and 80 ppm groups were 22.1, 21.8, 21.7 and 22.1 days.
Key result
Dose descriptor:
NOAEC
Remarks:
reproductive toxicity
Effect level:
>= 80 ppm (analytical)
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEC
Remarks:
parental systemic toxicity
Effect level:
5 ppm (analytical)
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
The numbers of F1 pups found dead and/or missing, as well as the general physical condition of all F1 pups in this study were unaffected by parental test article exposure.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
The mean number of pups born, live litter size, percentage of males per litter at birth and postnatal survival between birth and PND 0 (relative to number born), PND 0-1, 1-4 (pre-selection), 4 (post-selection)-7, 7-14, 14-21, and from birth to PND 4 (pre-selection) and PND 4 (post-selection)-28 were unaffected by the test article at all exposure concentrations. Differences from the control group were slight, not statistically significant and did not occur in an exposure-related manner.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean F1 male and female pup body weight and body weight gains in the 5, 20 and 80 ppm groups were similar to the control group during the period of suspended F0 maternal exposure (PND 1-4). Mean F1 male and female pup body weight gains in the 5 ppm group were decreased when compared to the control group during PND 4-7. However, there was no exposure-related relationship, and mean body weights in these pups were only slightly lower than those of the concurrent control group on PND 4 and 7. Mean male and female pup body weight gains in the 5, 20 and 80 ppm groups were slightly lower during PND 7-14, 14-21 and 21-28 when compared to the control group, but no exposure-related response was evident. Overall, statistically significant decreases in mean body weight gains were noted in the male and female pups in the 5, 20 and 80 ppm groups from PND 4 to 28. Mean body weights were statistically significantly decreased in the 5 ppm males on PND 14, in the 5 and 20 ppm males and 5 ppm females on PND 21 and in the 5, 20 and 80 ppm males and 5 and 80 ppm females on PND 28 when compared to the control group. By PND 28, mean male pup body weights in the 5, 20 and 80 ppm groups were 12.0%, 8.7% and 9.7% lower, respectively, than the control group, and mean female pup body weights were 9.8%, 7.3% and 9.1% lower in the same respective groups. However, there was no exposure-response relationship noted between the test article-exposed groups, and the 5 ppm group had the lightest body weights and the lowest overall weight gains.
Comparison of the F1 pup data with historical control values was confounded by the fact that the PND 4 pups in the test article-exposed groups had mean body weights slightly above the mean values in the WIL historical control data for inhalation studies, while the PND 28 values in these groups were slightly lower (7% or less) than historical control values. In addition, mean body weights in the concurrent control group were notably higher (4.2% to 5.3%) when compared to the mean values in the WIL historical control data for inhalation studies on PND 28. Examination of the mean F2 pup mean body weights and mean body weight gains from PND 1 through 28 did not reveal any statistically or biologically significant decreased values. In addition, when the F1 pup body weights in the 5, 20 and 80 ppm groups on PND 14, 21 and 28 were compared to the WIL historical control data for inhalation studies, only the PND 21 male and female body weights in the 5 ppm group were statistically significant. Given the lack of exposure response for decreases in pup body weight or weight gain in the F1 generation and the lack of a similar effect on pup weight in the F2 generation, the effects observed in the F1 generation were not considered related to exposure to the test article.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
* BALANOPREPUTIAL SEPARATION
Mean ages of attainment of balanopreputial separation were 47.7, 45.8 and 46.7 days in the 5, 20 and 80 ppm groups, respectively, compared to 44.9 days in the concurrent control group; the difference from control was statistically significant in the 5 ppm group and no exposure-response was observed. The mean value for the age of attainment of balanopreputial separation age in the WIL historical control data for inhalation studies was 46.3 days, suggesting that the differences observed in the 5 ppm group was not biologically significant and unrelated to test article exposure.
* VAGINAL PATENCY
Mean age at attainment of vaginal patency in the 5, 20 and 80 ppm groups was 36.2, 35.9 and 36.4 days, respectively, compared to 34.4 days in the concurrent control group. The differences in these same respective groups were statistically significant (p<0.05 or p<0.01) when compared to the concurrent control group; however no exposure-response relationship was observed. The mean value in the WIL historical control data for inhalation studies was 35.2 days with a range of 32.5 to 38.8 days and indicated that the differences noted in this study were unrelated to test article exposure.
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
- NECROPSIES OF WEANLINGS SELECTED FOR ORGAN WEIGHTS - PND 28
* ORGAN WEIGHTS
No effects on mean organ weights (absolute, relative to final body weight and relative to brain weight) were observed at any dosage level when the test article-exposed groups were compared to the control group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
- NECROPSIES OF PUPS FOUND DEAD OR EUTHANIZED IN EXTREMIS
The numbers of pups (litters) found dead or euthanized in extremis during PND 0-28 numbered 12(11), 16(12), 40(11) and 14(8) in the control, 5, 20 and 80 ppm groups, respectively. No internal findings that could be attributed to F0 parental exposure to the test article were noted in any group.
-NECROPSIES OF WEANLINGS NOT SELECTED FOR ORGAN WEIGHTS - PND 28
The numbers of pups (litters) examined viscerally on PND 28 numbered 106(29), 103(28), 102(27) and 99(27) in the control, 5, 20 and 80 ppm groups, respectively. No internal findings in any group were noted that could be attributed to F0 parental exposure to the test article.
- NECROPSIES OF WEANLINGS SELECTED FOR ORGAN WEIGHTS - PND 28
* MACROSCOPIC EXAMINATION
At the PND 28 necropsy of F1 weanlings selected for organ weights, internal findings included kidney cyst in a pup in the control group, adhesion on spleen (attached to the left lateral abdominal wall) in a pup in the 5 ppm group and dark red discoloration of the mandibular salivary gland in a pup and a small thymus in a pup in the 80 ppm group. These isolated findings were not attributed to test article exposure.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
80 ppm (analytical)
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
The numbers of F2 pups found dead and/or missing, as well as the general physical condition of all F2 pups in this study were unaffected by F1 parental test article exposure.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
The mean live litter size, percentage of males per litter at birth and postnatal survival between birth and PND 0 (relative to number born), PND 0-1, PND 1-4 (pre-selection), PND 4-7 (post-selection), PND 7-14, PND 14-21, PND 21-28, birth to PND 4 (preselection), and PND 4 (post-selection)-28 were unaffected by the test article at all exposure levels.
A female in the control group and a female in the 80 ppm group had total litter losses between PND 0-2.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean F2 pup body weight gain in test article-exposed groups was similar or slightly lower during the period of suspended F1 maternal exposure (PND 1-4) and following re-initiation of F1 maternal exposure (PND 4-7, 7-14, 14-21, 21-28 and 4-28) when compared to the concurrent control group values. Minor differences from control were not statistically significant (during PND 1-4 and 4-28), did not demonstrate an exposure-related relationship and were considered related to differences in live litter size between the control and test article-exposed groups. Mean F2 pup body weights were also similar or slightly lower (not statistically significant) during the entire lactation period when compared to the concurrent control group. The mean body weight values in the male and female concurrent control group were higher than the mean values in the WIL historical control data for inhalation studies at PND 1 (2.8% and 4.5%, respectively) and at PND 28 (5.3% and 7.0%, respectively). Therefore, slight differences noted in the test article-exposed groups compared to the concurrent control values were attributed to higher mean values in the concurrent control and were not considered biologically significant. In conclusion, mean pup body weight gains and body weights were unaffected at any exposure level throughout the lactation period.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test article-related effects on organ weights (absolute, relative to final body weight and relative to brain weight) were observed at any dosage concentration when the test article-exposed groups were compared to the control group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
- NECROPSIES OF PUPS FOUND DEAD
The numbers of pups (litters) found dead during PND 0 through the selection of the F2 generation numbered 9(7), 14(7), 9(7) and 13(8) in the control, 5, 20 and 80 ppm groups, respectively. No internal findings that could be attributed to parental exposure to the test article were noted at the necropsies of pups that were found dead.
- NECROPSIES OF WEANLINGS NOT SELECTED FOR ORGAN WEIGHTS - PND 28
At the PND 28 necropsy, no internal findings that could be attributed to F1 maternal test article exposure were noted at the necropsy of pups euthanized on PND 28.
- NECROPSIES OF WEANLINGS SELECTED FOR ORGAN WEIGHTS - PND 28
At the PND 28 necropsy of F2 weanlings selected for organ weights, internal findings included cyst(s) on right kidneys in 1 pup in each of the control and 5 ppm groups, dilated pelvis in the kidney in 1 pup in each of the 5 and 80 ppm groups and dark red areas in the thymus in 1 pup in each of the control, 5 ppm and 80 ppm groups. These findings were not considered test article-related because they occurred randomly in single pups.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEC
Generation:
F2
Effect level:
80 ppm (analytical)
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Text Table 1: Organ weight changes associated with body weight changes at 80 ppm in F0 generation animals

Organ

Direction of change a

Sex

Brain (relative to body weight)

increase

Male, Female

Cauda, epididymis, right (relative to body weight)

increase

Male

Epididymis, left (absolute)

¯

Male

Epididymis, left (relative to body weight)

increase

Male

Epididymis, right (relative to body weight)

increase

Male

Testis, left (relative to body weight)

increase

Male

Testis, right (relative to body weight)

increase

Male

Kidneys (absolute)

decrease

Male

Kidneys (relative to brain weight)

decrease

Male

Liver (absolute)

decrease

Male

Liver (relative to brain weight)

decrease

Male

Spleen (absolute)

decrease

Male

Spleen (relative to brain weight)

decrease

Male

Adrenal glands (relative to body weight)

increase

Female

Adrenal glands (relative to brain weight)

increase

Female

Final Body Weightb

decrease

Male, Female

a = The indication of higher or lower refers to a statistically significant difference between test article-exposed and control group animals.

b = Final body weights in the 80 ppm males and females (491 g and 324 g, respectively) were significantly different from the control group (571 g and 350 g for males and females, respectively) at 0.01 using Dunnett's test.

Text Table 2 : Organ weight changes associated with body weight changes in F1 generation animals

Organ

Direction of change(a

Sex

Exposure level (ppm)

Brain (absolute)

decrease

Female

Male

80

20, 80

Brain (relative to body weight)

increase

Female

80

Cauda, epididymis, left (absolute)

decrease

Male

80

Cauda, epididymis, left (relative to body weight)

increase

Male

20

Cauda, epididymis, right (relative to bodyweight)

increase

Male

20, 80

Epididymis, left (relative to body weight)

increase

Male

20, 80

Epididymis, right (absolute)

decrease

Male

80

Epididymis, right (relative to body weight)

increase

Male

20, 80

Testis, left (absolute)

decrease

Male

80

Testis, left (relative to body weight)

increase

Male

80

Testis, right (absolute)

decrease

Male

5, 20, 80

Testis, right (relative to body weight)

increase

Male

80

Kidneys (absolute)

decrease

Male

Female

20, 80

80

Kidneys (relative to brain weight)

decrease

Male, Female

80

Liver (absolute)

decrease

Male

Female

20, 80

80

Liver (relative to brain weight)

decrease

Male, Female

80

Spleen (absolute)

decrease

Male, Female

80

Spleen (relative to brain weight)

decrease

Male

80

Adrenal glands (relative to body weight)

increase

Male, Female

80

Adrenal glands (relative to brain weight)

increase

Female

80

Pituitary (absolute)

decrease

Male

5, 20, 80

Pituitary (relative to brain weight

decrease

Male

5, 20, 80

Prostate (relative to body weight)

increase

Male

80

Seminal vesicles/coagulating glands/fluid (absolute)

decrease

Male

80

Final Body Weight (b

decrease

decrease

Male

Female

5, 20, 80

20, 80

a = The indication of higher or lower refers to a statistically significant difference between test article-exposed and control group animals.

b = Final body weights in the males in the 20 and 80 ppm groups (549 g and 522 g, respectively) and in the females in the 80 ppm group (294 g) were significantly different from the control group (614 g and 340 g for males and females, respectively) at 0.01 using Dunnett’s test. Final body weights in the males in the 5 ppm group and females in the 20 ppm group (574 g and 329 g, respectively) were not statistically different.

TABLE 3: F1 - PRE-WEANING - SUMMARY OF OFFSPRING WEIGHTS [G] (LITTER AS EXPERIMENTAL UNIT)

GROUP:

SEX

0 PPM

5 PPM

20 PPM

80 PPM

PND 1

MALES

MEAN

7.2

7.1

7.1

7.1

S.D.

0.73

0.71

0.78

0.60

N

29

28

29

27

FEMALES

MEAN

6.7

6.7

6.7

6.7

S.D.

0.58

0.76

0.66

0.54

N

29

28

29

27

PND 4 (BEFORE SELECTION)

MALES

MEAN

10.1

9.8

10.2

10.1

S.D.

1.47

1.37

1.27

1.11

N

29

28

28

27

FEMALES

MEAN

9.3

9.4

9.6

9.6

S.D.

1.26

1.42

1.13

1.05

N

29

28

28

27

PND 7

MALES

MEAN

13.8

12.7

13.3

13.5

S.D.

2.28

1.80

1.96

1.97

N

29

28

28

27

FEMALES

MEAN

12.8

12.1

12.6

12.7

S.D.

2.18

2.02

1.82

1.87

N

29

28

28

27

PND 14

MALES

MEAN

26.4

23.8*

24.5

24.7

S.D.

3.69

3.71

3.65

2.75

N

29

28

28

27

FEMALES

MEAN

24.9

22.9

23.5

23.4

S.D.

3.92

3.94

3.49

2.48

N

29

28

28

27

PND 21

MALES

MEAN

42.0

36.4**

38.2*

37.9

S.D.

6.47

6.66

6.30

5.79

N

29

28

28

27

FEMALES

MEAN

39.6

35.1*

36.7

36.2

S.D.

6.10

6.50

6.15

5.28

N

29

28

28

27

PND 28

MALES

MEAN

79.7

70.1**

72.8*

72.0*

S.D.

10.

66 11.

70 10.

94 9.88

N

29

28

28

27

FEMALES

MEAN

72.7

65.6*

67.4

66.1*

S.D.

9.99

10.58

9.

67 8.36

N

29

28

28

27

PND = POSTNATAL DAY

* = SIGNIFICANTLY DIFFERENT FROM THE CONTROL GROUP AT 0.05 USING COVARIATE ANALYSIS

** = SIGNIFICANTLY DIFFERENT FROM THE CONTROL GROUP AT 0.01 USING COVARIATE ANALYSIS

Conclusions:
In a reproduction study, four groups of male and female Crl:CD(SD) rats (30/sex/group) were exposed to either clean filtered air or vapor atmospheres of DMDS for 6 hours daily over 2 generations The exposure concentrations were 0, 5, 20 and 80ppm. The first parental generation (F0) was exposed for at least 70 consecutive days prior to mating. General systemic toxicity was seen in the 20 and 80 ppm F0 and F1 parental males and females characterized by decreased mean body weights. body weight gains and/or food consumption. The NOAEL for parental systemic toxicity was 5 ppm. No functional effects on reproduction or on pups were seen at any tested concentration. The NOAEL for reproductive and developmental toxicity was 80 ppm.
Executive summary:

In a 2 -generation reprotoxicity study performed following the OECD guideline # 416, four groups of male and female Crl:CD) SD) rats (30/sex/group) were exposed to either clean filtered air or vapor atmospheres of the test article, dimethyl disulfide, for 6 hours daily for at least 70 consecutive days prior to mating at DMDS concentrations of 0, 5, 20 and 80 ppm for the F0 and F1 generations. The first parental generation F0 animals were approximately 6.6 weeks of age at the beginning of exposure. The offspring selected to become the F1 parental generation were exposed following weaning (beginning on postnatal day [PND] 28). Exposure of the F0 and F1 males continued throughout mating and through the day prior to euthanasia. The F0 and F1 females continued to be exposed throughout mating and gestation through gestation day 20. After parturition, exposure of the F0 and F1 females was re-initiated on lactation day 5 and continued through the day prior to euthanasia. F0 animals were exposed for 142 consecutive days, and F1 animals weree exposed for 158 consecutive days. All animals were observed twice daily for appearance and behavior. Clinical observations, body weights and food consumption weree recorded at appropriate intervals for males throughout the study and for females prior to mating and during gestation and lactation. Vaginal lavage was performed daily to determine estrous cycles beginning 21 days prior to pairing. All F0 and F1 females were allowed to deliver and rear their pups until weaning on lactation day 28. Clinical observations, body weights and sexes for F1 and F2 pups were recorded at appropriate intervals. For both generations (F1 and F2), 8 pups per litter (4 per sex, when possible) were selected on PND 4 to reduce the variability among the litters. Offspring (30/sex/goup) from the pairing of the F0 animals were selected by PND 21 to constitute the F1 generation. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the selected F1 rats. Nonseleeted F1 and F2 weanlings were necropsied on PND 28. Selected organs were weighed from both F1 and F2 pups that were necropsied on PND 28. Each surviving F0 and F1 parental animal received a complete detailed gross necropsy following the completion of weaning of the F1 and F2 pups, respectively; selected organs were weighted. Spermatogenic endpoints (sperm motility including progressive motility, morphology and numbers) were recorded for all surviving F0 and F1 males, and ovarian primordial follicle counts were recorded for all surviving F0 and F1 females, in the control and high-exposure groups. Designated tissues from all F0 and F1 parental animals were weighed and examined microscopically.

There were no functional effects on reproduction (estrous cycles, mating and fertility indices, number of days between pairing and coitus, and gestation length) in any test article-exposed group. There were no adverse effects on pups born to dams exposed to the test article and results from several studies confirm a lack of effect on postnatal growth prior to weaning with exposure of the lactating dams. Therefore, in this study an exposure level of 80 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive and developmental toxicity when the test article was administered via whole-body inhalation exposure to Crl:CD(SD) rats. General systemic toxicity was evident in the 20 and 80 ppm group F0 and F1 parental males and females with persistent decrements in mean body weights, body weight gains and/or food consumption. Potential exposure-related effects on the adrenal glands (an increase in the incidence of vacuolization of the adrenal cortex or increased adrenal gland weights [relative to final body weight or brain weight]) were noted in the F0 and F1 parental animals in the 80 ppm group. The F0 and F1 5 ppm parental groups were not affected by these parameters other than slight transitory decreases in body weight gain and/or food consumption. Therefore, a NOAEL for parental systemic toxicity was considered to be 5 ppm.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1995
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina,
- Age at reception: 58 days old
- Body weight at exposure initiation: (P) Males: 342 - 392 g; Females: 219 - 274 g
- Housing (non exposure periods): individually in wire-mesh cages (except during the exposure period) suspended above cage-board.
- Diet : PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002, ad libitum. Food was withheld during the exposure periods.
- Water: municipal water, ad libitum. Water was withheld during the exposure periods.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS (NON-EXPOSURE PERIODS)
- Temperature (°C): 22 ± 3 (actual : 21.2°C to 21.7°C)
- Humidity (%): 50 ± 20 (actual: 36.8% to 47.8%)
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2005-02-22 To: 2005-06-14
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
Exposures were conducted in four 1.0-m3 stainless steel and glass exposure chambers. One chamber was dedicated to each of the 3 test article exposure groups and the 4th chamber was dedicated to the control group. The exposure period was 6 hours per day, 7 days per week and was defined as the time between turning the generation system on and off, including the start equilibration time. The chambers were operated under dynamic conditions, at a slight negative pressure (ca 0.5 in of water) with at least 12 to 15 air changes per hour.
Details on mating procedure:
The animals were paired on a 1:1 basis within each exposure group following 14 days of exposure for the males and females. A breeding record containing the male and female identification numbers and the start date of cohabitation was prepared. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Actual exposure concentrations within each chamber were measured at least 10 times (approximately every 35 minutes) during each daily exposure period by a validated gas chromatographic method. Exposure atmosphere samples were delivered to the gas chromatograph from the approximate middle of each chamber by a pump and multi-port sampling valve. At least 1 standard was analyzed each day prior to exposure to confirm gas chromatographic calibration. Overall mean measured test article exposure concentrations for the F0 generation were 0, 5, 50 and 150 ppm and for the F1 generation were 0, 5.1, 50 and 150 ppm for the filtered air, 5, 50 and 150 ppm groups, respectively.
Duration of treatment / exposure:
The F0 males and females were exposed to the test atmosphere for 14 consecutive days prior to mating. Exposure of the F0 males continued throughout mating and through the day prior to euthanasia. The F0 females continued to be exposed throughout mating through gestation day 20; exposure was re-initiated on lactation day 5 and continued through lactation day 27. During lactation, the dams were removed from their litters during each daily 6-hour exposure period. One F1 pup/sex/litter was selected for inhalation exposure beginning on PND 28 (following weaning) and continuing until PND 34.
Frequency of treatment:
6 hours per day, 7 days per week
Remarks:
Doses / Concentrations:
5, 50 and 150 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
12
Control animals:
other: The control group was exposed to clean, filtered air under conditions identical to those used for the groups exposed to test article.
Positive control:
Not appropriate
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
All F0 rats were observed twice daily, once in the morning and once in the afternoon, for appearance, behavior, moribundity and mortality. During inhalation exposures, animals that were visible through the chamber windows were observed for appearance and behavior at the mid-point of exposure. In addition, each animal was observed for signs of toxicity within 1 hour following exposure. All significant findings were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
Individual detailed clinical observations were recorded weekly.

BODY WEIGHT: Yes
- Individual male body weights were recorded on the first day of exposure, weekly thereafter and at termination.
- Individual female body weights were recorded on the first day of exposure, weekly thereafter until evidence of copulation was observed. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 1, 4, 7, 14, 21 and 28.

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

WATER CONSUMPTION : No
Oestrous cyclicity (parental animals):
Not done.
Sperm parameters (parental animals):
Not done
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

F1 LITTER PARAMETERS
LITTER VIABILITY AND DEATHS
Each litter was examined daily for survival, and all deaths were recorded.
CLINICAL OBSERVATIONS
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1, 4, 7, 14, 21 and 28.
BODY WEIGHTS
Pups were individually weighed on PND 1, 4, 7, 14, 21 and 28.
SEX DETERMINATION
Pups were individually sexed on PND 0, 1, 4, 14 and 28.
WEANING AND SELECTION
Each dam and litter remained housed together until weaning on lactation day 28. One male and 1 female F1 weanling from each group (control, 5, 50 and 150 ppm) were randomly selected using a computerized randomization procedure prior to weaning (PND 28) to comprise the F1 generation. These pups (a minimum of 1 male and 1 female per litter, when available) were exposed to the control or test article for 6 hours per day beginning on day 28 post partum.
Postmortem examinations (parental animals):
F0 MACROSCOPIC EXAMINATIONS
Males were euthanized following completion of the mating period. Females that delivered were euthanized on lactation day 28 and females with total litter loss were euthanized within 24 hours of litter loss; the numbers of former implantation sites were recorded.
Necropsy included examination of the external surface, all orifices and the cranial cavity, the external surface of the brain, and the thoracic, abdominal and pelvic cavities, including viscera. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.

ORGAN WEIGHTS
The following organs were weighed from all F0 animals at the scheduled necropsies:
Brain Ovaries
Epidiymidesa Pituitary
Kidneys Testes(a
Liver
(a = These paired organs were weighed separately.

MICROSCOPIC EXAMINATIONS
Microscopic examination was performed on the following tissues from all animals in the control and 150 ppm groups at the scheduled necropsies and from the animal euthanized in extremis during the study.
Cervix, Coagulating gland, Epididymides, Mammary glands, Ovaries (2), Pituitary gland, Prostate gland, Seminal vesicles (2), Testes, Vas deferens, Uterus, Vagina, All gross (internal) lesions
Postmortem examinations (offspring):
EUTHANASIA OF F1 GENERATION
F1 pups not selected for exposure were euthanized via isoflurane inhalation on PND 28 and examined internally for gross abnormalities.F1
weanlings selected for exposure were euthanized via carbon dioxide inhalation on PND 35 and discarded without macroscopic examination.
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 by sex. Where applicable, the litter was used as the experimental unit. Parental mating, fertility, conception and copulation indices were analyzed using the Chi-square test with Yates’ correction factor. Mean parental body weights (weekly, gestation and lactation), body weight changes and food consumption, offspring body weights and body weight changes, gestation length,
numbers of implantation sites, number of pups born, live litter size on PND 0, unaccounted-for sites, absolute and relative organ weights, pre-coital intervals values 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 was used to compare the test article-exposed groups to the control group. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA 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. Mean litter proportions
(percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test article-exposed groups to the control group. Histopathological findings in the test article-exposed groups were compared to the control group using a two-tailed Fisher’s
Exact test.
Reproductive indices:
Mating, fertility and copulation, conception indices were calculated.
Offspring viability indices:
The following litter parameters were calculated: Mean Live Litter Size, Postnatal Survival Between Birth and PND 0 or PND 4, Postnatal Survival for All Other Intervals
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One F0 female in the 150 ppm group was euthanized in extremis on lactation day 7 following a body weight loss of 39 g. This female had several clinical findings prior death, including a pale body and eyes, shallow respiration and red material around the nose and mouth. A cause of death could not be determined at necropsy or microscopically (with a limited number of tissues being examined). However, the moribund condition of this female was considered test article-related since body weight effects were noted for other animals in this group and spontaneous mortality is low in rats, especially during lactation. All other animals survived to the scheduled necropsies. No test article-related clinical findings were observed in the F0 males and surviving F0 females.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Effects on mean body weights, body weight changes and food consumption were noted in the 50 and 150 ppm group F0 males throughout the study and in the 150 ppm group F0 females during gestation and lactation. During the first week of the premating period (study week 0-1), a mean body weight loss was noted at 150 ppm, while a reduced mean body weight gain was noted at 50 ppm compared to the control group. Mean body weight gains in the 150 ppm group were reduced for the remainder of the study (study weeks 2-3 and 3-4) while mean body weight gains in the 50 ppm group were reduced during study week 3-4. As a result, mean body weights in the 50 and 150 ppm group F0 males were up to 5.5% and 12.6% less than the control group, respectively, during study weeks 1-4. Mean F0 maternal body weight gains were reduced during most of the gestation period, and during lactation days 4-7 (following resumption of test article exposure). Mean body weights in this group were up to 12.5% and 10.0% lower than the control group during the gestation and lactation periods, respectively. Mean F0 maternal food consumption was generally reduced in the 150 ppm group females throughout gestation and lactation.
There were no effects on body weights, body weight gains or food consumption in F0 males at 5 ppm and in F0 females at 5 and 50 ppm. Mean body weights in the 50 ppm group F0 females were lower on lactation days 1 and 7. The slight reductions (approximately 6%) were attributed to the reduced mean body weight gains noted during the premating period and were not considered evidence of toxicity during lactation.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
One F0 female in the 5 ppm group had total litter loss on lactation day 1. No total litter loss occurred at 50 or 150 ppm; therefore, the occurrence
at 5 ppm was not considered test article-related.
Reproductive performance (mating, fertility, copulation and conception indices) was unaffected by exposure to the test article. The mean numbers of days between pairing and coitus and mean gestation lengths were unaffected by test article exposure. There were no signs of dystocia in this study.
A slight reduction in the mean number of implantation sites in the 150 ppm group was attributed to a single female with an atypically low value (4 implantation sites); no relationship to the test article was evident. The mean number of implantation sites in the 5 and 50 ppm groups and the mean number of unaccounted for implantation sites in the 5, 50 and 150 ppm groups were similar to the control group value.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No test article-related effects on organ weights were noted in the F0 males or females. Any differences from the control group were slight, did not occur in an exposure-related manner, or were explained by reduced mean body weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no test article-related macroscopic findings in F0 males and
females.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no test article-related microscopic findings in F0 males and
females.
Dose descriptor:
NOAEC
Remarks:
parental systemic toxicity
Effect level:
5 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: Decrements in body weight gain and food consumption in the 50 (males only) and 150 ppm groups.
Dose descriptor:
NOAEC
Remarks:
parental reproductive toxicity
Effect level:
150 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: No functional effects on reproduction at any exposure concentration.
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
n/a
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
VIABILITY (OFFSPRING)
The mean number of pups born and live litter size on PND 0 were slightly lower in the 150 ppm group as a result of a single female, which also had an atypically low number of implantation sites. No relationship to the test article was evident. No test article-related effects on the mean number of pups born, live litter size and the percentage of males at birth were observed in the 5 and 50 ppm groups. Postnatal survival in the 150 ppm group was lower during PND 4-28 due to a single female that lost 5 pups during PND 5-9. There were no effects of maternal exposure to the test article on postnatal survival in the 5 and 50 ppm groups.

CLINICAL SIGNS (OFFSPRING)
The general physical condition of the F1 pups was not affected by maternal exposure to the test article. There were no clinical findings in the F1 pups selected for 1 week of direct test article exposure during the post-weaning period.

BODY WEIGHT (OFFSPRING)
Mean F1 male and female
pup body weight gains were lower in the 50 and 150 ppm groups during PND 4-28 (females) and PND 7-14 (males). As a result, mean body weights in the 50 and 150 ppm groups were up to 15.3% and 17.0% less (females) and up to 11.2% and 6.7% less (males) than the control group, respectively. Mean body weights and body weight gains of the F1 pups selected for direct test article exposure were further reduced. Mean body weights in the 50 and 150 ppm groups were 18.3% and 17.6% (males) and 18.8% and 21.4% (females) lower than the control group on PND 35, respectively. There were no effects of maternal exposure (pre-weaning) or direct exposure (post-weaning) on mean body weights or body weight gains in the 5 ppm group F1 males and females.

GROSS PATHOLOGY (OFFSPRING)
There were no macroscopic findings in F1 pups that were found dead, euthanized due to the death of the dam or at the scheduled necropsies that could be attributed to maternal test article exposure (pre-weaning) or direct exposure to the test article (post-weaning) at any exposure level.
Dose descriptor:
NOAEC
Remarks:
neonatal toxicity
Generation:
F1
Effect level:
5 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: Reduced F1 pup body weights and body weight gains.
Critical effects observed:
no
Reproductive effects observed:
not specified

Table 1: Group mean body weights (g) Males

Study time point

 

0 ppm

5 ppm

50 ppm

150 ppm

0

Mean

SD

N

368

13.6

12

368

12.4

12

367

12.1

12

365

14.4

12

1

Mean

SD

N

389

15.8

12

386

16.2

12

371

17.6

12

357**

25.2

12

2

Mean

SD

N

403

18.9

12

400

18.7

12

381

21.7

12

363**

26.9

12

3

Mean

SD

N

419

17.8

12

419

19.7

12

400

25.9

12

376**

29.6

12

4

Mean

SD

N

446

23.7

12

446

23.0

12

424

27.7

12

390**

34.3

12

** significantly different from control at 0.01 using Dunnett’s test

 

Table2: Group mean body weight changes (g) Males

Study time point

 

0 ppm

5 ppm

50 ppm

150 ppm

0-1

Mean

SD

N

22

6.1

12

18

7.4

12

4**

12.9

12

-8**

13.6

12

1-2

Mean

SD

N

13

7.9

12

14

5.7

12

10

7.9

12

7*

5.6

12

2-3

Mean

SD

N

17

6.4

12

19

7.8

12

19

6.5

12

13

5.8

12

3-4

Mean

SD

N

27

6.5

12

27

7.4

12

23

3.9

12

14**

6.5

12

* significantly different from control at 0.05 using Dunnett’s test

** significantly different from control at 0.01 using Dunnett’s test

 

Table 3: Group mean body weights (g) Females

Study time point

 

0 ppm

5 ppm

50 ppm

150 ppm

Week 0

Mean

SD

N

241

7.8

12

243

8.7

12

242

13.0

12

239

11.4

12

Week 1

Mean

SD

N

255

7.9

12

250

8.9

12

245

11.7

12

247

10.5

12

Week 2

Mean

SD

N

262

12.5

12

257

13.0

12

249

14.5

12

252

9.6

12

GD 0

Mean

SD

N

264

12.1

11

257

14.1

10

256

15.9

11

251

11.1

11

GD 4

Mean

SD

N

286

11.6

11

282

13.7

10

277

17.6

12

270

10.1

11

GD 7

Mean

SD

N

297

12.3

11

291

14.2

10

286

18.2

12

275**

10.1

11

GD 11

Mean

SD

N

318

11.6

11

309

17.4

10

305

20.9

12

293**

11.6

11

GD 14

Mean

SD

N

333

12.5

11

324

20.8

10

319

20.3

12

307**

12.0

11

GD 17

Mean

SD

N

362

13.6

11

347

33.5

10

348

20.4

12

327**

13.7

11

GD 20

Mean

SD

N

408

15.0

11

387

49.4

10

393

25.3

12

357**

19.6

11

** significantly different from control at 0.01 using Dunnett’s test

GD – gestation day

 

Table 4: Group mean body weight changes (g) Females

Study time point

 

0 ppm

5 ppm

50 ppm

150 ppm

Week 0-1

Mean

SD

N

14

7.9

12

7

8.8

12

3*

10.1

12

8

10.0

12

Week 1-2

Mean

SD

N

7

10.1

12

7

6.9

12

4

5.9

12

5

5.1

12

Week 0-2

Mean

SD

N

21

10.4

12

14

9.0

12

7**

9.6

12

13

11.1

12

GD 0-4

Mean

SD

N

22

7.1

11

25

6.7

10

22

8.1

11

19

5.7

11

GD 4-7

Mean

SD

N

12

2.8

11

10

4.2

10

9

3.8

12

5

12.5

11

GD 7-11

Mean

SD

N

21

4.1

11

18

5.3

10

19

5.1

12

19

14.3

11

GD 11-14

Mean

SD

N

15

4.7

11

15

4.9

10

14

4.8

12

14

2.1

11

GD 14-17

Mean

SD

N

29

5.5

11

23

15.0

10

28

3.9

12

20*

5.3

11

GD 17-20

Mean

SD

N

46

4.4

11

41

17.1

10

45

7.2

12

30*

7.3

11

* significantly different from control at 0.05 using Dunnett’s test

 

Table 5: Group mean body weights (g) Females

Study time point

 

0 ppm

5 ppm

50 ppm

150 ppm

LD 1-A

Mean

SD

N

310

17.4

11

298

16.5

9

291*

18.3

12

284**

12.0

11

LD 4-A

Mean

SD

N

321

18.3

11

313

19.7

8

308

23.7

12

301

13.3

11

LD 7

Mean

SD

N

339

13.4

11

326

18.4

8

318*

21.0

12

305**

19.9

11

LD 14

Mean

SD

N

358

17.0

11

346

18.6

8

345

17.3

12

327**

18.4

10

LD 21

Mean

SD

N

359

9.8

11

356

20.3

8

357

20.9

12

334**

18.2

10

LD 28

Mean

SD

N

313

11.8

11

309

18.0

8

316

14.3

12

311

14.9

10

* significantly different from control at 0.05 using Dunnett’s test

** significantly different from control at 0.01 using Dunnett’s test

LD – lactation day (A - non-exposed)

 

Table 6: Group mean body weight changes (g) Females

Study time point

 

0 ppm

5 ppm

50 ppm

150 ppm

LD 1-4A

Mean

SD

N

11

12.0

22

17

13.2

8

17

9.9

12

18

8.5

11

LD 4-7

Mean

SD

N

29

14.2

11

13

7.4

8

10

8.4

12

4

16.4

11

LD 7-14

Mean

SD

N

19

11.4

11

20

10.8

8

27

9.4

12

18

7.6

10

LD 14-21

Mean

SD

N

1

11.9

11

10

15.5

8

13

10.2

12

7

9.4

10

LD 21-28

Mean

SD

N

-46

9.0

11

-47

14.1

8

-41

14.8

12

-23**

11.6

10

LD 1-28

Mean

SD

N

3

14.0

11

14

10.4

8

25**

13.2

12

28**

14.8

10

 

Table 7: Summary of food consumption (g/rat/day)

Time point

 

0 ppm

5 ppm

50 ppm

150 ppm

MALES

Week 0-1

Mean

SD

n

26

1.5

12

25

2.0

12

23**

2.1

12

20**

2.6

12

Week 1-2

Mean

SD

N

25

1.5

12

24

2.2

12

23

2.4

12

21**

2.2

12

FEMALES

Week 0-1

Mean

SD

n

18

1.2

12

18

1.1

12

16*

2.0

12

16**

1.7

12

Week 1-2

Mean

SD

N

19

1.4

12

18

1.3

12

17

1.8

12

17*

1.6

12

* significantly different from the control group at 0.05 using Dunnett’s test

** significantly different from the control group at 0.01 using Dunnett’s test

Food consumption was not recorded during the breeding period (weeks 2 to 4)

 

Table 8: Summary of food consumption in Females (g/rat/day)

Time point

 

0 ppm

5 ppm

50 ppm

150 ppm

GD 0-4

Mean

SD

n

20

2.3

11

20

2.1

10

19

2.2

12

17*

2.1

11

GD 4-7

Mean

SD

N

22

2.2

11

21

3.3

10

22

2.1

12

19**

1.7

11

GD 7-11

Mean

SD

n

22

2.0

11

20

1.0

10

21

2.6

12

19**

1.8

11

GD 11-14

Mean

SD

N

23

2.3

11

21

2.2

10

23

2.6

12

20

2.3

11

GD 14-17

Mean

SD

N

25

3.0

11

22

4.7

10

24

2.5

12

21*

2.1

11

GD 17-20

Mean

SD

N

26

2.1

11

23

4.6

10

25

2.2

12

22**

2.1

11

GD 0-20

Mean

SD

N

23

1.9

11

21

2.7

10

22

1.8

12

19**

1.8

11

* significantly different from the control group at 0.05 using Dunnett’s test

** significantly different from the control group at 0.01 using Dunnett’s test

GD gestation day

 

Table 9: Summary of food consumption in Females (g/rat/day)

Time point

 

0 ppm

5 ppm

50 ppm

150 ppm

LD 1-4A

Mean

SD

n

35

7.4

11

38

8.3

8

38

6.7

12

36

6.7

9

LD 4-7

Mean

SD

N

45

4.6

11

44

7.2

8

43

4.9

12

36**

8.0

11

LD 7-14

Mean

SD

n

54

4.0

11

53

10.8

8

51

7.6

12

45*

7.0

10

LD 14-21

Mean

SD

N

65

4.5

11

62

13.9

8

64

7.5

12

57

9.4

10

LD 21-28

Mean

SD

N

113

13.2

11

101

29.2

8

106

16.1

12

97

22.4

10

LD 1-28

Mean

SD

N

69

4.0

11

65

15.1

8

66

8.3

12

62

10.1

8

* significantly different from the control group at 0.05 using Dunnett’s test

** significantly different from the control group at 0.01 using Dunnett’s tes

LD lactation day

A non exposure period

Conclusions:
There were no functional effects on reproduction (mating and fertility indices, number of days between pairing and coitus, and gestation length) at any exposure concentration. Therefore, an exposure level of 150 ppm was considered to be the NOAEL (no-observed-adverse-effect level) for parental reproductive toxicity of DMDS when administered via whole-body inhalation exposure to rats. In general, evidence of general toxicity was more pronounced in the F0 males than in the F0 females and consisted of decrements in body weight gain and food consumption in the 50 (males only) and 150 ppm groups. Therefore, the NOAEL for parental systemic toxicity was considered to be 5 ppm. Neonatal toxicity was expressed at 50 and 150 ppm by reduced F1 pup body weights and body weight gains. Mean F1 male and female body weights and body weight gains were reduced further after 1 week of direct DMDS exposure during the postweaning period. Therefore, the NOAEL for neonatal toxicity was 5 ppm.
Executive summary:

This study was designed to provide preliminary information on the potential adverse effects of DMDS on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behaviour, conception, parturition and lactation of the F0generation and the development of offspring from conception through day 28 of postnatal life.

Four groups of male and female Crl:CD(SD) rats (12/sex/group) were exposed to either clean filtered air or vapour atmospheres of DMDS, for 6 hours daily for 14 consecutive days prior to mating. DMDS concentrations were 0, 5, 50 and 150 ppm.

Exposure of the F0males continued during the mating period and through the day prior to euthanasia for a total of 29 days of exposure. The F0females continued to be exposed throughout mating and gestation through gestation day 20. After parturition, exposure of the F0females was re-initiated on lactation day 5 and continued through the day prior to euthanasia (lactation day 28). The F0females that delivered were exposed for a total of 58-72 days. Females with evidence of mating that failed to deliver were exposed through post-mating day 20 for a total of 35-49 days, and then euthanized on post-mating day 25.

The F1offspring (1/sex/litter) selected for control or DMDS exposure were exposed following weaning, beginning on postnatal day (PND) 28 through PND 34. DMDS concentrations were 0, 5, 50 and 150 ppm.

All animals were observed twice daily for appearance and behaviour. Clinical observations, body weights and food consumption were recorded weekly for F0males throughout the study and for F0females prior to mating. During gestation and lactation these parameters were recorded at appropriate intervals. All F0females were allowed to deliver and rear their pups until lactation day 28. Litters were culled to 10 pups/litter (5/sex, when possible) on PND 4 to reduce the variability among the litters. F1clinical observations and body weights were recorded on PND 1, 4, 7, 14, 21 and 28 for all pups and also on PND 35 for pups selected for exposure. F1pups not selected for exposure were necropsied on PND 28, and exposed F1weanlings were euthanized and discarded without examination on PND 35. F0males were euthanized following completion of the mating period and F0females were euthanized on lactation day 28. Complete necropsies were conducted on all F0animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0animals in the control and high-exposure groups.

 

There was one DMDS related early death in the 150 ppm F0female group on lactation day 7. This female was euthanized following a body weight loss of 39 g and several clinical findings prior death, including a pale body and eyes, shallow respiration and red material around the nose and mouth. A cause of death could not be determined at necropsy or microscopically (with a limited number of tissues being examined).

One F0female in the 5 ppm group had total litter loss on lactation day 1. No total litter loss occurred at 50 or 150 ppm; therefore, the occurrence at 5 ppm was not considered DMDS related. No DMDS related clinical findings were observed in the F0males and surviving F0females. Reproductive performance (mating, fertility, copulation and conception indices), mean numbers of days between pairing and coitus and mean gestation lengths was unaffected by DMDS exposure. There were no signs of dystocia in this study.

Effects on mean body weights, body weight changes and food consumption were noted in the 50 and 150 ppm group F0males throughout the study and in the 150 ppm group F0females during gestation and lactation. During the first week of the premating period, a mean body weight loss was noted at 150 ppm, while a reduced mean body weight gain was noted at 50 ppm compared to the control group. Mean body weight gains in the 150 ppm group were reduced for study weeks 2-3 and 3-4 while mean body weight gains in the 50 ppm group were reduced during study week 3-4. Mean F0maternal body weight gains were reduced during most of the gestation period, and during lactation days 4-7 (following resumption of DMDS exposure).

Mean F0maternal food consumption was generally reduced in the 150 ppm group females throughout gestation and lactation. There were no effects on body weights, body weight gains or food consumption in F0males at 5 ppm and in F0females at 5 and 50 ppm. Mean body weights in the 50 ppm F0females were lower on lactation days 1 and 7 which was attributed to the reduced mean body weight gains noted during the premating period and not considered evidence of toxicity during lactation. There were no DMDS related macroscopic or microscopic findings in F0males and females. No DMDS related effect on the number of implantation sites was identified and there were no DMDS effects on organ weights noted in the F0males or females.

The mean number of pups born and live litter size on PND 0 were slightly lower in the 150 ppm group as a result of a single female, which also had an atypically low number of implantation sites; no relationship to DMDS was evident. No DMDS related effects on the mean number of pups born, live litter size and the percentage of males at birth were observed in the 5 and 50 ppm groups. Postnatal survival in the 150 ppm group was lower during PND 4-28 due to a single female that lost 5 pups during PND 5-9. There were no effects of maternal exposure to DMDS on postnatal survival in the 5 and 50 ppm groups. The general physical condition of the F1pups was not affected by maternal exposure to DMDS.

There were no clinical findings in the F1pups selected for 1 week of direct DMDS exposure during the post-weaning period. Mean F1male and female pup body weight gains were lower in the 50 and 150 ppm groups during PND 4-28 (females) and PND 7-14 (males). Mean body weights and body weight gains of the F1pups selected for direct DMDS exposure were further reduced. There were no effects of maternal exposure (pre-weaning) or direct exposure (post-weaning) on mean body weights or body weight gains in the 5 ppm group F1males and females. There were no macroscopic findings in F1pups that were found dead, euthanized due to the death of the dam or at the scheduled necropsies that could be attributed to maternal DMDS exposure (pre-weaning) or direct exposure to DMDS (post-weaning) at any exposure level.

There were no functional effects on reproduction (mating and fertility indices, number of days between pairing and coitus, and gestation length) at any exposure concentration. Therefore, an exposure level of 150 ppm was considered to be the NOAEL (no-observed-adverse-effect level) for parental reproductive toxicity of DMDS when administered via whole-body inhalation exposure to rats. In general, evidence of general toxicity was more pronounced in the F0males than in the F0females and consisted of decrements in body weight gain and food consumption in the 50 (males only) and 150 ppm groups. Therefore, the NOAEL for parental systemic toxicity was considered to be 5 ppm. Neonatal toxicity was expressed at 50 and 150 ppm by reduced F1pup body weights and body weight gains. Mean F1male and female body weights and body weight gains were reduced further after 1 week of direct DMDS exposure during the postweaning period. Therefore, the NOAEL for neonatal toxicity was 5 ppm.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
308 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
GLP guideline study
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

2-generation reproduction study (2006)


A 2-generation reproduction study in rats was conducted to evaluate the potential adverse effects of dimethyl disulphide (DMDS) on male and female reproductive capabilities, including gonadal function, oestrous cyclicity, mating behaviour, conception, gestation, parturition, lactation and weaning of the F0 and F1 generations and F1 and F2 neonatal survival, growth and development (Nemec, 2006b). One litter per dam was produced in each generation. There were no functional effects on reproduction (oestrous cycles, mating and fertility indices, number of days between pairing and coitus, and gestation length) at any DMDS-exposed groups. There were no adverse effects on pups born to dams exposed to DMDS and results from several studies confirm a lack of effect on postnatal growth prior to weaning with exposure of the lactating dams. Therefore, in this study an exposure level of 80 ppm was considered to be the NOAEC for reproductive and developmental toxicity when DMDS was administered via whole-body inhalation to Sprague-Dawley rats. General systemic toxicity was evident in the 20 and 80 ppm group F0 and F1 parental males and females with persistent decrements in mean body weights, body weight gains and/or food consumption. Potential exposure-related effects on the adrenal glands (an increase in the incidence of vacuolization of the adrenal cortex or increased adrenal gland weights [relative to final body weight and brain weight]) were noted in the F0 and F1 parental animals in the 80 ppm group. At 5 ppm, The F0 and F1 parental groups were not affected showed slight transitory decreases in body weight gain and/or food consumption that were not considered adverse. Therefore, the NOAEC for parental toxicity was considered to be 5 ppm.
In a Reproduction / Developmental Toxicity Screening Test performed according to the OECD Guideline #421, 4 groups of male and female Crl: CD-SD) rats (12/sex/group) were exposed to either clean filtered air or vapor atmospheres of dimethyl disulphide (DMDS), for 6 hours daily for 14 consecutive days prior to mating (Nemec, 2006c). Target and measured test article concentrations were 0, 5, 50 and 150 parts per million (ppm). Exposure of the F0 males continued during the mating period and through the day prior to euthanasia for a total of 29 days of exposure. The F0 females continued to be exposed throughout mating and gestation through gestation day 20. After parturition, exposure of the F0 females was re-initiated on lactation day 5 and continued through the day prior to euthanasia. There were no functional effects on reproduction (mating and fertility indices, number of days between pairing and coitus, and gestation length) at any exposure concentration. Therefore, an exposure level of 150 ppm was considered to be the NOAEL (no-observed-adverse-effect level) for parental reproductive toxicity of DMDS when administered via whole-body inhalation exposure to rats. In general, evidence of general toxicity was more pronounced in the F0 males than in the F0 females and consisted of decrements in body weight gain and food consumption in the 50 (males only) and 150 ppm groups. Therefore, the NOAEL for parental systemic toxicity was considered to be 5 ppm. Neonatal toxicity was expressed at 50 and 150 ppm by reduced F1 pup body weights and body weight gains. Mean F1 male and female body weights and body weight gains were reduced further after 1 week of direct test article exposure during the postweaning period. Therefore, the NOAEL for neonatal toxicity was 5 ppm.


 


Reproduction screening (2006)


This study was designed to provide preliminary information on the potential adverse effects of DMDS on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behaviour, conception, parturition and lactation of the F0generation and the development of offspring from conception through day 28 of postnatal life.


Four groups of male and female Crl:CD(SD) rats (12/sex/group) were exposed to either clean filtered air or vapour atmospheres of DMDS, for 6 hours daily for 14 consecutive days prior to mating. DMDS concentrations were 0, 5, 50 and 150 ppm.


Exposure of the F0 males continued during the mating period and through the day prior to euthanasia for a total of 29 days of exposure. The F0 females continued to be exposed throughout mating and gestation through gestation day 20. After parturition, exposure of the F0 females was re-initiated on lactation day 5 and continued through the day prior to euthanasia (lactation day 28). The F0females that delivered were exposed for a total of 58-72 days. Females with evidence of mating that failed to deliver were exposed through post-mating day 20 for a total of 35-49 days, and then euthanized on post-mating day 25.


The F1 offspring (1/sex/litter) selected for control or DMDS exposure were exposed following weaning, beginning on postnatal day (PND) 28 through PND 34. DMDS concentrations were 0, 5, 50 and 150 ppm.


All animals were observed twice daily for appearance and behaviour. Clinical observations, body weights and food consumption were recorded weekly for F0males throughout the study and for F0females prior to mating. During gestation and lactation these parameters were recorded at appropriate intervals. All F0females were allowed to deliver and rear their pups until lactation day 28. Litters were culled to 10 pups/litter (5/sex, when possible) on PND 4 to reduce the variability among the litters. F1clinical observations and body weights were recorded on PND 1, 4, 7, 14, 21 and 28 for all pups and also on PND 35 for pups selected for exposure. F1pups not selected for exposure were necropsied on PND 28, and exposed F1weanlings were euthanized and discarded without examination on PND 35. F0males were euthanized following completion of the mating period and F0females were euthanized on lactation day 28. Complete necropsies were conducted on all F0animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0animals in the control and high-exposure groups.


 


There was one DMDS related early death in the 150 ppm F0 female group on lactation day 7. This female was euthanized following a body weight loss of 39 g and several clinical findings prior death, including a pale body and eyes, shallow respiration and red material around the nose and mouth. A cause of death could not be determined at necropsy or microscopically (with a limited number of tissues being examined).


One F0female in the 5 ppm group had total litter loss on lactation day 1. No total litter loss occurred at 50 or 150 ppm; therefore, the occurrence at 5 ppm was not considered DMDS related. No DMDS related clinical findings were observed in the F0males and surviving F0females. Reproductive performance (mating, fertility, copulation and conception indices), mean numbers of days between pairing and coitus and mean gestation lengths was unaffected by DMDS exposure. There were no signs of dystocia in this study.


Effects on mean body weights, body weight changes and food consumption were noted in the 50 and 150 ppm group F0males throughout the study and in the 150 ppm group F0females during gestation and lactation. During the first week of the premating period, a mean body weight loss was noted at 150 ppm, while a reduced mean body weight gain was noted at 50 ppm compared to the control group. Mean body weight gains in the 150 ppm group were reduced for study weeks 2-3 and 3-4 while mean body weight gains in the 50 ppm group were reduced during study week 3-4. Mean F0maternal body weight gains were reduced during most of the gestation period, and during lactation days 4-7 (following resumption of DMDS exposure).


Mean F0maternal food consumption was generally reduced in the 150 ppm group females throughout gestation and lactation. There were no effects on body weights, body weight gains or food consumption in F0males at 5 ppm and in F0females at 5 and 50 ppm. Mean body weights in the 50 ppm F0females were lower on lactation days 1 and 7 which was attributed to the reduced mean body weight gains noted during the premating period and not considered evidence of toxicity during lactation. There were no DMDS related macroscopic or microscopic findings in F0males and females. No DMDS related effect on the number of implantation sites was identified and there were no DMDS effects on organ weights noted in the F0males or females.


The mean number of pups born and live litter size on PND 0 were slightly lower in the 150 ppm group as a result of a single female, which also had an atypically low number of implantation sites; no relationship to DMDS was evident. No DMDS related effects on the mean number of pups born, live litter size and the percentage of males at birth were observed in the 5 and 50 ppm groups. Postnatal survival in the 150 ppm group was lower during PND 4-28 due to a single female that lost 5 pups during PND 5-9. There were no effects of maternal exposure to DMDS on postnatal survival in the 5 and 50 ppm groups. The general physical condition of the F1pups was not affected by maternal exposure to DMDS.


There were no clinical findings in the F1pups selected for 1 week of direct DMDS exposure during the post-weaning period. Mean F1male and female pup body weight gains were lower in the 50 and 150 ppm groups during PND 4-28 (females) and PND 7-14 (males). Mean body weights and body weight gains of the F1pups selected for direct DMDS exposure were further reduced. There were no effects of maternal exposure (pre-weaning) or direct exposure (post-weaning) on mean body weights or body weight gains in the 5 ppm group F1males and females. There were no macroscopic findings in F1pups that were found dead, euthanized due to the death of the dam or at the scheduled necropsies that could be attributed to maternal DMDS exposure (pre-weaning) or direct exposure to DMDS (post-weaning) at any exposure level.


There were no functional effects on reproduction (mating and fertility indices, number of days between pairing and coitus, and gestation length) at any exposure concentration. Therefore, an exposure level of 150 ppm was considered to be the NOAEL (no-observed-adverse-effect level) for parental reproductive toxicity of DMDS when administered via whole-body inhalation exposure to rats. In general, evidence of general toxicity was more pronounced in the F0males than in the F0females and consisted of decrements in body weight gain and food consumption in the 50 (males only) and 150 ppm groups. Therefore, the NOAEL for parental systemic toxicity was considered to be 5 ppm. Neonatal toxicity was expressed at 50 and 150 ppm by reduced F1pup body weights and body weight gains. Mean F1male and female body weights and body weight gains were reduced further after 1 week of direct DMDS exposure during the postweaning period. Therefore, the NOAEL for neonatal toxicity was 5 ppm.

Effects on developmental toxicity

Description of key information

GLP- and recognised test guideline-compliant inhalation developmental toxicity studies in the rat (Barker, 1991 and Nemec, 2006d) and rabbit (Nemec, 2005a) are available for dimethyl disulphide. In the rat, the NOAEL for maternal toxicity was 20 ppm, the NOAEL for teratogenicity was 80 ppm (the highest dose level tested and examined), and the NOAEL for embryotoxicity was 20 ppm. In the rabbit, the NOAEL for maternal toxicity was 135 ppm, the NOAEL for teratogenicity was 135 ppm (the highest dose level tested), and the NOAEL for embryotoxicity was 135 ppm.

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Limit test:
no
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
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
Dose / conc.:
5 ppm
Dose / conc.:
20 ppm
Dose / conc.:
80 ppm
No. of animals per sex per dose:
27
Control animals:
yes, sham-exposed
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
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
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
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
Abnormalities:
no effects observed
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.
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
Abnormalities:
no effects observed
Developmental effects observed:
no
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.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
May 1989 - January 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
1981
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ORGANISMS:
- Source: Charles River UK Ltd., Margate
- Age at reception: 10-12 weeks - Weight at reception: 165-218 g
- Number of animals:  100 rats : 25 females / dose group (3 dose groups +  1 control group)
- Acclimatation period: no data

HOUSING
The animals were housed in group of 6 in suspended stainless steel cages.

FOOD and WATER
- Food: SQC rat and Mouse breeder Diet No. 3 ad libitum excepted during  exposure
- Water: filtered tap water, ad libitum excepted during exposure

ENVIRONMENTAL CONDITIONS
- Temperature : 19-25°C
- Relative humidity : 40-70%
- Light/dark cycle : 12h/12h
- Ventilation : 15 air changes/hour
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
- Production of test atmospheres:
Four horizontal flow, recirculating exposure chambers were used. Each was  made of stainless steel with perspex (Plexiglas) doors and a fan to mix  the atmospheres by recirculation. The compressed air supply was from a  clean, dry, filtered source. The 3 concentrations of test article vapour  were produced by passing metered flows of air through sintered glass  frits immersed in separate containers of test article. The resulting  outputs of vapour were introduced to the diluent air inlet duct of each  test chamber. Mixing, within the duct and recirculation system, ensured  the production of homogeneous atmospheres for animal exposure. The  chambers were ventilated at a rate of at least 12 air changes per hour.  Air flows were monitored continuously and recorded twice hourly during  exposure. The exhaust streams were purified with activated charcoal and  vented to the outside of the building.
- Vehicle: filtered air
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Exposure chamber test article concentration
* Measured concentration
Samples for analysis were withdrawn from the exposure chambers twice  hourly through sample lines leading from each chamber through a sampling  valve into a total hydrocarbon analyser. The analysis was performed with an Analysis Automation total hydrocarbon  analyser type 523 Detector with a Flame ionisation detector (FID) . The chamber concentrations of the test article were close to target  values throughout the exposure period.

* Nominal concentration
The total weight of test article used and total volume of diluent air  were measured for each exposure.
- Group  /  Target  /  Nominal  /  Analytical concentrations      
1         /        0     
2         /        5     /       9.27    /        5.17 ppm     
3         /        15    /    26.71    /        15.35 ppm     
4         /        50    /    86.59    /        50.46 ppm
Duration of treatment / exposure:
day 6 to day 15 of gestation
Frequency of treatment:
6 h/day
Duration of test:
up to gestation day 20
Dose / conc.:
5 ppm
Dose / conc.:
15 ppm
Dose / conc.:
50 ppm
No. of animals per sex per dose:
30
Control animals:
yes, sham-exposed
Details on study design:
Three groups of 30 mated female rats were exposed to DMDS by whole body exposure at 5, 15 or 50 ppm for 6 hours daily from day 6 to day 15 of gestation. A similar group of 30 rats, exposed to filtered air only over the same period, served as controls. All animals were maintained until day 20 of gestation, killed and their uterine content assessed.
Maternal examinations:
- Morbidity and mortality 
All females were examined twice daily to detect any which  were dead or moribund.
- Clinical observations All females were examined daily from day 3 to day 20 of  gestation. Any abnormalities of appearance or behaviour or  other signs of reaction to treatment or ill health were  recorded.
- Body weight The body weight of each female was recorded on days 3, 6, 10, 15, and 20  of gestation.
- Food intake The amount of food consumed by each cage of females was recorded daily  from day 3 to day 20 of gestation and reported on the body weight  intervals.
- Terminal studies
* Necropsy  All females were killed on day 20 of gestation, in random group order, by  cervical dislocation and examined macroscopically.
Ovaries and uterine content:
The ovaries and uteri were removed and examined and the following data  recorded: pregnancy status number of corpora lutea number and intrauterine position of implantations  subdivided into: live foetuses, early intrauterine deaths,  late intrauterine deaths and dead foetuses.
Fetal examinations:
Live foetuses were killed by an intracardiac injection of sodium  pentobarbitone solution (200 mg/ml). Foetuses were weighed individually, examined externally and sexed.  Approximately one half of the foetuses in each litter (selected by  systematic sampling) were dissected and the viscera were examined. They  were then eviscerated and the carcasses processed to stain the ossified  skeleton (Alizarin technique). The skeleton was examined and preserved  and stored in absolute glycerol (containing thymol crystals). The remaining foetuses were placed in Bouin's fluid for at least two  weeks to allow fixation and partial decalcification. They were then  transferred to 70% industrial methylated spirit. At examination, the head  was removed by a coronal cut through the mouth, pharynx and back of the  head and sections of the head were examined. The remaining portion of the  foetus was examined by dissection and was preserved, with the head  sections, in Bouin's fluid and stored in plastic vials. Foetal abnormalities were recorded as malformations (rare and/or  potentially lethal defects) and variations (cormnonly occurring  non-lethal abnormalities).
Statistics:
The following parameters were analysed:
Body weight, body weight gain, food intake, litter weight : analysis of variance, Dunnett's test
Fertilitiy indices: Cochran-Armitage test and Fisher-Irwin exact test with Bonferroni adjustment
Corpora lutea, implantations, foetuses, pre-implantation loss and post-implantation loss, malformation, foetal weight, percentage male foetuses: Kruskal-Wallis non-parametric Anonva, Wilcoxon's rank-sum test and Terpstra-Jonkcheere test.
malformations
Indices:
Percentage pre-implantation loss, percentage post-implantation loss and percentage male foetuses
Historical control data:
Available.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
A higher  incidence of rough haircoat was observed at 50 ppm. Clinical condition at  5 and 15 ppm did not differ from controls. 
Description (incidence and severity):
n/a
Mortality:
no mortality observed
Description (incidence):
There were no deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Dosage-related reductions in  weight gain were observed at 15 and 50 ppm. At 50 ppm, weight gain was 40% lower than control over the exposure period (day 6 to 15, p < 0.001). At 15 ppm, weight gain over the exposure period was 16% lower than control group days 6 to 15, p< 0.01)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake was lower (p< 0.001) than  controls at 50 ppm but comparable at 5 or 15 ppm.
Description (incidence and severity):
n/a
Water consumption and compound intake (if drinking water study):
not examined
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):
No unusual lesions were observed at necropsy. 
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
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
Changes in pregnancy duration:
not examined
Description (incidence and severity):
n/a
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Pregnancy rates were lower  than expected (73.3, 50.0, 86.7 and 70.0% at 0, 5, 15 and 50 ppm, respectively), but sufficient data was available in all groups for adequate assessment of the study results.
Dose descriptor:
NOAEC
Remarks:
Maternal toxicity
Effect level:
5 ppm (analytical)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced at 50 ppm. At 5  and 15 ppm these parameters were comparable to controls. 
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
Smaller than expected in all groups (12.0 to 14.0).
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Reduced at 50 ppm. At 5  and 15 ppm these parameters were comparable to controls. 
Anogenital distance of all rodent fetuses:
not examined
Description (incidence and severity):
n/a
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
A slightly higher  incidence of retarded ossification was observed at 50 ppm but was  considered to indicate delayed maturation, as a result of the lower  foetal weight, rather than a teratogenic effect.
No malformations.
Visceral malformations:
no effects observed
Dose descriptor:
NOAEC
Remarks:
Developmental toxicity
Effect level:
15 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
changes in litter size and weights
Abnormalities:
no effects observed
Developmental effects observed:
no

 Table 1: Summary of body weight and changes during gestation (g)

Day of gestation

0 ppm

5 ppm

15 ppm

50 ppm

3

209

209

206

208

6

231

232

228

226

10

260

262

251**

241***

15

297

297

283**

265***

20

351

360

346

327

% weight change

days 3-20

67.9

72.2

68.0

57.2

% weight change

days 6-15

28.6

28.0

24.1**

17.3***

** body weight gain significantly lower than control days 6-10, 6-15 (p<0.01 analysis of variance, Dunnett's test)

*** body weight gain significantly lower than control days 6-10, 6-15, 10-15 (p<0.001 analysis of variance, Dunnett's test)

 

Table 2: Summary of food intake (g/animal/day)

Days of gestation

0 ppm

5 ppm

15 ppm

50 ppm

3-6

25

25

26

25

6-10

25

24

24

21***

10-15

26

25

25

21***

15-20

28

27

30

27

Mean intake (g/day) days 3-20

26

25

26

23

Mean intake (g/day) days 6-15

26

25

25

21***

*** significantly lower than control (p<0.001, analysis of variance, Dunnett's test)

 

Table 3 : Summary of uterine/implantation data

Group mean caesarian data

0ppm

5 ppm

15 ppm

50 ppm

Number of females with live foetuses at day 20 gestation

 

22

 

15

 

26

 

21

Number of corpora lutea

299

215

353

289

Mean number per female

13.6

14.3

13.6

13.8

Number of implantations

264

174

310

244

Mean number per female

12.0

11.6

11.9

11.6

% pre-implantation loss

11.7

19.1

12.2

15.6

Number of early intrauterine deaths

5

12

13

10

Mean number per female

0.2

0.8

0.5

0.5

Number of late intrauterine deaths

1

0

2

0

Mean number per female

0.0

0.0

0.1

0.0

Number of dead foetuses

0

0

0

0

Mean number per female

0.0

0.0

0.0

0.0

%post-implantation loss

2.3

6.9

4.8

4.1

Number of foetuses

258

162

295

234

Mean number per female

11.7

10.8

11.3

11.1

% of implantations

97.7

93.1

95.2

95.9

 

Table 4: Summary of number of foetuses and litter weights (g)

 

0 ppm

5 ppm

15 ppm

50 ppm

Number of male foetuses

128

73

152

119

Number of female foetuses

130

89

143

115

%male foetuses

49.6

45.1

51.5

50.9

Mean litter weight

43.7

41.8

41.7

38.8

Mean foetal weight

3.8

3.9

3.7

3.5**

Mean foetal weight males only

4.0

4.1

3.8

3.6**

Mean foetal weight females only

3.7

3.7

3.6

3.4**

** significantly different from control at 0.01 by non-parametric ANOVA and Wilcoxon rank-sum test

Table 5:foetal defect data

 

0 ppm

5 ppm

15 ppm

50 ppm

EXTERNAL AND VISCERAL DEFECTS

Number of foetuses examined

258

162

295

234

Number showing malformations

1

0

0

0

% of foetuses examined

0.4

0.0

0.0

0.0

Number showing variations

52

27

65

52

% of foetuses examined

20.2

16.7

22.0

22.2

SKELETAL DEFECTS

Number of foetuses examined

136

85

155

121

Number showing malformations

1

0

0

0

% of foetuses examined

0.7

0.0

0.0

0.0

Number showing variations

123

74

139

116

% of foetuses examined

90.4

87.1

89.7

95.9

Total number of foetuses showing malformations

1

0

0

0

% of foetuses examined

0.4

0.0

0.0

0.0

 

 

Conclusions:
Exposure to DMDS at 50 ppm elicited maternal toxicity, with associated foetal growth retardation (demonstrated by low weight and retarded ossification). There was no indication of a teratogenic effect. At 15 ppm, less marked maternal toxicity was observed and there were no foetal effects. There was no adverse effect of treatment, maternal or foetal, at 5 ppm.
Executive summary:

In a developmental toxicity study, four groups of 30 mated Crl:CD(SD)BR female rats were exposed to either clean filtered or DMDS vapor atmospheres of 5, 15 or 50 ppm for 6 hours daily in whole-body inhalation chambers during gestation days 6 through 15. 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 uteri, placentae and ovaries were examined and the pregnancy status, number of corpora lutta and the number and intrauterine position of implantations of live and dead fetuses and early and late resorptions were recorded. The fetuses were weighed, sexed and examined. Approximately one half of the fetuses from each litter were dissected and the viscera examined. The carcasses were prossed for skeletal examination following staining with Alizarin red. The remaining fetuses were placed in Bouins solution and evaluated for soft tissue abnormalities and developmental variations. The study authors reported that the maternal LOAEC was 15 ppm based on reduced body weight gain. The NOAEC for maternal toxicity was reported to be 5 ppm. The fetal/developmental toxicity LOAEC, was 50 ppm based on lover mean fetal weight and retarded ossification. The NOAEC for fetal developmental toxicity was 15 ppm.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June - September 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products, Inc., Denver, Pennsylvania
- Age at reception: 5.5 months old
- Gestation age at reception: 1, 2 or 3 days
- Weight at study initiation: 3063 g to 4625 g for the animals in replicate one and 3049 g to 4150 g for animals in replicate two on gestation day 0
- Housing: individually in clean, stainless steel wire-mesh cages suspended above ground corncob bedding
- Diet: PMI Nutrition International, LLC, Certified Rabbit LabDiet® 5322, ad libitum
- Water: Reverse osmosis-purified (on-site) drinking water, ad libitum
- Acclimation period: 3-5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18.7°C to 23.5°C
- Humidity: 49.2% to 63.8%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
The exposures were conducted (in two sequential replicates) in four 2.0 m3 stainless steel and glass whole-body exposure chambers. One chamber was dedicated for each group for the duration of the study. Food and water were withheld during each daily exposure period.
Vapors of the test article were generated using glass gas washing bottles contained individually in a Plexiglas cube under slight negative pressure. Nitrogen from a regulated in-house source and monitored by Omega and Cole-Parmer rotameters, flowed through the fritted disc of the gas washing bottle. Concentrated vapors of the test article were then carried to the chamber inlet and mixed 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 of the test article atmospheres were determined by gas chromatography (GC). Samples of the exposure atmospheres were automatically collected at approximately 35-minute intervals using a sample loop and a computer-controlled multiposition valve.
The overall mean concentrations for the first replicate were 15.3 ppm for the 15 ppm group, 45.0 ppm for the 45 ppm group, and 135.0 ppm for the 135 ppm group. The overall mean concentrations for the second replicate were 15.0 ppm for the 15 ppm group, 45.0 ppm for the 45 ppm group, and 134.6 ppm for the 135 ppm group.
Duration of treatment / exposure:
Gestation days 6-28 (23 exposures)
Frequency of treatment:
6 hours per day, 7 days per week
Duration of test:
until sacrifice on GD 29
Dose / conc.:
15 ppm
Dose / conc.:
45 ppm
Dose / conc.:
135 ppm
No. of animals per sex per dose:
24
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale:
Exposure levels were selected based on results of a range-finding prenatal developmental toxicity study of dimethyl disulfide in rabbits (WIL-160117, 2005). In the main range-finding phase of that study (Phase II) no maternal or developmental toxicity was observed at exposure levels of 1, 5, 25 and 50 ppm. Due to the lack of maternal and developmental toxicity in that phase, an additional 5–day toxicity study was conducted (Phase III) in nonpregnant females at the exposure levels of 75 and 150 ppm of DMDS. Based on the clinical signs (including head bobbing, head held high and eyes partially closed), body weight losses generally throughout the study and sustained reductions in ood consumption in the 150 ppm group for 5 consecutive days, the high exposure level of 135 ppm in the current study was selected because it was expected to induce maternal toxicity.
Maternal examinations:
CLINICAL OBSERVATIONS AND SURVIVAL
All rabbits were observed twice daily, once in the morning and once in the afternoon (at least 7 hours apart) for moribundity and mortality. Individual detailed clinical observations were recorded from gestation days 0 through 29 (prior to exposure during the treatment period). Animals were also observed for signs of toxicity at the midpoint of exposure and approximately 1 hour following exposure. Additional observations were recorded at the beginning (approximately 20-30 minutes) of exposure when necessary (3 days).

BODY WEIGHTS
Individual maternal body weights were recorded on gestation days 0 (by the supplier under non-GLP conditions), 4 and 6-29 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 6-10, 10-14, 14-21, 21-29, 6-21, 6-29 and 0-29.

GRAVID UTERINE WEIGHTS
Gravid uterine weight was collected and net body weight (the day 29 body weight exclusive of the weight of the uterus and contents) and net body weight change (the day 0-29 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

FOOD CONSUMPTION
Individual food consumption was recorded on gestation days 6-29 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals.
Ovaries and uterine content:
FEMALE THAT ABORTED
The female that aborted in this study was euthanized by an intravenous injection of sodium pentorbital via a marginal ear vein that day and necropsied. The number and location of implantation sites, corpora lutea and the number of viable fetuses were recorded. Gross lesions were saved for possible future histopathologic examination.

GESTATION DAY 29 LAPAROHYSTERECTOMY
All surviving rabbits were euthanized on gestation day 29 by an intravenous injection of sodium pentobarbital via the marginal ear vein. The thoracic, abdominal and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined.
The uterus and ovaries were then exposed and excised. 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. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.
Fetal examinations:
FETAL MORPHOLOGICAL EXAMINATION
Each viable fetus was examined externally and individually weighed. The detailed external examination of each fetus included, but was not limited to,
an examination of the eyes and palate and external orifices.Crown-rump measurements and degrees of autolysis were recorded for late resorptions.
Each viable fetus was subjected to a visceral examination using a modification of the fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was determined by internal examination. Fetal kidneys were examined and graded for renal papillae development. Heads from all fetuses were examined by a mid-coronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol.
Following fixation in alcohol, each fetus was macerated in potassium hydroxide and stained with Alizarin Red S (Dawson, 1926). External, visceral and skeletal findings were recorded as developmental variations or malformations.
Statistics:
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 was used to compare the test article-treated 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, the Mann-Whitney U-test was used to compare the test article-treated groups to the control group.
Historical control data:
Available.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical observations noted at the beginning (approximately 20-30 minutes) of the 6-hour exposure period included head held high in two and four animals in the 45 and 135 ppm groups, respectively, and wet clear material around the mouth in three animals in the 135 ppm group on the first day of exposure only. However, these findings were not sustained with continuation of exposure and therefore were not considered adverse effects of the test article.
Description (incidence and severity):
n/a
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights, body weight gains, net body weights, net body weight gains and gravid uterine weights in the 15, 45 and 135 ppm groups were unaffected by test article exposure throughout gestation (days 6-10, 10-14, 14-21, 21-29 and 6-29).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A statistically significant (p<0.05) decrease in food consumption (evaluated as g/animal/day and g/kg/day) was noted in the 135 ppm group on the first day of exposure (gestation day 6-7) when compared to the control group. The reduction in food consumption was considered test article-related, as food consumption in the 135 ppm group remained slightly lower (not statistically significant) than the control group throughout the first 2 weeks of exposure (gestation days 6-10, 10-14, 14-21 and 6-21), but was not considered adverse because of the lack of concomitant effects on mean body weights and body weight gains. During gestation days 21-29 food consumption in the 135 ppm group was similar to the control group. Food consumption in the 15 and 45 ppm groups was unaffected by exposure.
Description (incidence and severity):
n/a
Water consumption and compound intake (if drinking water study):
not examined
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:
effects observed, treatment-related
Description (incidence and severity):
One, three, six (including the female that aborted) and six females in the control, 15, 45 and 135 ppm groups, respectively, had dark red discoloration of or dark red areas on the lungs (generally all lobes).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
One, three, six (including the female that aborted) and six females in the control, 15, 45 and 135 ppm groups, respectively, had dark red discoloration of or dark red areas on the lungs (generally all lobes).
Histopathological findings: neoplastic:
not examined
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One female the 45 ppm group aborted on gestation day 26.
Dose descriptor:
NOAEC
Remarks:
maternal toxicity
Effect level:
>= 135 ppm (analytical)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
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
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
There were no external malformations or developmental variations noted in the 15, 45
and 135 ppm groups. In the control group, two fetuses had umbilical herniation of the
intestine (several loops of the intestine protruded through an opening in the umbilicus).
Skeletal malformations:
no effects observed
Description (incidence and severity):
Skeletal malformations were observed in 6(3), 3(3) and 2(2) fetuses (litters) in the
control, 15 and 45 ppm groups, respectively. Two, two and one fetuses in the control, 15
and 45 ppm groups, respectively, had vertebral anomalies with or without associated rib
anomalies consisting of absent and extra arches, centra and/or ribs, mislocated centra and
fused ribs and centra. Rib anomalies consisting of extra, fused or forked ribs were noted
for three and one fetuses in the control and 45 ppm groups, respectively. One fetus each
in the 15 and 45 ppm groups had skull anomalies consisting of medially fused nasal or
frontal bones. One control group fetus had a costal cartilage anomaly (bifurcated right
costal cartilage with the posterior fork associating with the sternum, causing subsequent
costal cartilages to associate with the sternum higher than normal). None of the
proportional values were statistically significant compared to the control group and the
values were within the WIL historical control data ranges. No skeletal malformations
were noted at the 135 ppm exposure level and none occurred at a higher incidence than in
the control group; therefore, the skeletal malformations were not considered
exposure-related.
The percent per litter of 13th full ribs in the 45 and 135 ppm groups (44.8% and 55.5%
per litter, respectively) were increased compared to the control group (31.3% per litter).
Although the difference was statistically significant (p<0.05) for the 135 ppm group, the
values were within the WIL historical control data range (19.4% - 59.1% per litter) and in
the absence of other indicators of developmental toxicity was not considered related to exposure. Additionally, the percent per litter value of 7th cervical ribs in the 45 ppm
group (7.9% per litter) exceeded the maximum mean value in the WIL historical control
data (7.7% per litter); however, this increase did not occur in an exposure-related manner
(3.1% per litter in the 135 ppm group) and was not considered exposure-related. Other
skeletal developmental variations occurred in all groups, including the control group, and
consisted of sternebra(e) nos. 5 and/or 6 unossified, bent hyoid arches, 13th rudimentary
rib(s), accessory skull bones and 27 presacral vertebrae. The mean litter percent of the
skeletal variants observed in the exposure groups occurred similarly to the control group
or were within the range of the WIL historical control data. No relationship to test article
exposure was evident.
Visceral malformations:
no effects observed
Description (incidence and severity):
Soft tissue malformations were observed in 3(3), 2(1), 3(1) and 3(2) fetuses (litters) in the
control, 15, 45 and 135 ppm groups, respectively. One, one, three and two fetuses in the
same respective groups had lobular agenesis of the lungs (absent right accessory lobe).
One fetus each in the control, 15 and 135 ppm groups had hydrocephaly (increased
cavitation of both the lateral and third ventricles). One fetus in the control group had
lobular dysgenesis in the lungs (all lobes small; right lobes fused). These findings
occurred similarly in the control group and the proportional values were within the range
of WIL historical control data, and were not considered exposure-related.
Soft tissue developmental variations occurred in all groups, including the control group,
and consisted primarily of blood vessel variations (the left carotid artery arose from the
brachiocephalic trunk, right subclavian artery coursed retroesophageal rejoined aortic
arch adjacent to ductus arteriosis with no brachiocephalic trunk, right carotid and
subclavian arteries arose independently from the aortic arch with no brachiocephalic
trunk), accessory spleens, retrocaval ureter, and small or absent gallbladder. Other soft tissue developmental variations observed in the test article-exposed groups occurred
infrequently, occurred similarly in the control group or the values were within the range
of WIL historical control data, therefore no test article-related effects were evident.
Fetus no. 40113-04 in the control group had cysts on the liver and clear fluid contents in
the thoracic and abdominal cavities. These findings were not classified as either
malformations or developmental variations and were not included in any tabulation.
Key result
Dose descriptor:
NOAEC
Remarks:
Developmental toxicity
Effect level:
>= 135 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
Transient clinical observations were noted at 45 and 135 ppm on the first day of exposure only and decreased food consumption at 135 ppm was sustained throughout the first 2 weeks of exposure but not considered adverse. Additionally, a macroscopic finding of dark red discoloration of or dark red areas on the lungs (generally all lobes) was noted at all exposure levels. No developmental toxicity was observed in any fetuses in this study.
Therefore, an exposure level of 135 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity (based on the lack of adverse effects on maternal body weight gain, food consumption and survival) and an exposure level of 135 ppm was considered to be the NOAEL for developmental toxicity when dimethyl disulfide was administered via whole-body inhalation to rabbits and developmental toxicity when dimethyl disulfide was administered via whole-body inhalation to rabbits.
Executive summary:

Dimethyl disulphide (DMDS) was evaluated in an inhalation prenatal developmental toxicity study in rabbits performed following the OECD guidleine # 414. DMDS was administered via whole-body inhalation as a vapor to three groups of 24 time-mated female New Zeeland White rabbits on a 6-hours per day basis during gestation days 6 through 28. A concurrent control group of 24 time-mated rabbits were exposed to clean, filtered air on a comparable regimen. The DMDS exposure concentrations were 0, 15, 45 and 135 ppm.

Transient clinical observations were noted at 45 and 135 ppm on the first day of exposure only and decreased food consumption at 135 ppm was sustained throughout the first 2 weeks of exposure but in the absence of effects on maternal body weight gains these findings were not considered adverse. Food consumption in the 15 and 45 ppm groups and mean body weights, body weight gains, net body weights, net body weight gains and gravid uterine weights in the 15, 45 and 135 ppm groups were unaffected by DMDS exposure for the duration of the study. A macroscopic finding of dark red discoloration of or dark red areas on the lungs (generally all lobes) was noted at all exposure levels. Intrauterine growth and survival was unaffected by test article exposure and no test article related malformations or developmental variations were noted at any exposure level.

An exposure level of 135 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity (based on the lack of adverse effects on maternal body weight gain, food consumption and survival) and an exposure level of 135 ppm was considered to be the NOAEL for developmental toxicity when dimethyl disulfide was administered via whole-body inhalation to rabbits.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
77 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
GLP guideline studies
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental toxicity study in rats


In a key 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 dimethyl disulphide (DMDS) for 6 hours daily in whole-body inhalation chambers during gestation days 6 through 19 (Nemec, 2006d). 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 calculated. The fetuses were 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 fetal/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.



In an earlier OECD 414 study, three groups of 30 mated female rats were exposed to DMDS by whole body exposure at 0, 5, 15 or 50 ppm for 6 hours daily from day 6 to 15 of gestation (Barker, 1991). All animals were killed on day 20 of gestation, and their uterine contents assessed. There were no deaths. A higher incidence of rough hair coat was observed at 50 ppm. Reductions in weight gain were observed at 15 and 50 ppm and food intake was reduced at 50 ppm. Litter and fetal weights were reduced at 50 ppm. No malformations were observed in fetuses from the treated groups. A slightly higher incidence of retarded ossification was observed at 50 ppm. Exposure to DMDS at 50 ppm elicited maternal toxicity with associated fetal growth retardation, which was demonstrated by low weight and retarded ossification. The NOAEL was 5 ppm for maternal toxicity, and 15 ppm for embryofetal effects (Barker, 1991).


 


Developmental toxicity study in rabbits


Dimethyl disulphide (DMDS) was evaluated in an inhalation prenatal developmental toxicity study in rabbits performed following the OECD guidleine # 414 (Nemec, 2005a). DMDS was administered via whole-body inhalation as a vapor to three groups of 24 time-mated female New Zeeland White rabbits on a 6-hoursper day basis during gestation days 6 through 28. A concurrent control group of 24 time-mated rabbits were exposed to clean, filtered air on a comparable regimen. The DMDS exposure concentrations were 0, 15, 45 and 135 ppm. Transient clinical observations were noted at 45 and 135 ppm on the first day of exposure only and decreased food consumption at 135 ppm was sustained throughout the first 2 weeks of exposure but in the absence of effects on maternal body weight gains these findings were not considered adverse. Food consumption in the 15 and 45 ppm groups and mean body weights, body weight gains, net body weights, net body weight gains and gravid uterine weights in the 15, 45 and 135 ppm groups were unaffected by DMDS exposure for the duration of the study. A macroscopic finding of dark red discoloration of or dark red areas on the lungs (generally all lobes) was noted at all exposure levels. Intrauterine growth and survival was unaffected by test article exposure and no test article related malformations or developmental variations were noted at any exposure level. An exposure level of 135 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity (based on the lack of adverse effects on maternal body weight gain, food consumption and survival) and an exposure level of 135 ppm was considered to be the NOAEL for developmental toxicity when dimethyl disulphide was administered via whole-body inhalation to rabbits.

Toxicity to reproduction: other studies

Description of key information

In a study (Nemec, 2006e) conducted in order to determine if the pup body weight effects noted in the concurrent two-generation study (Nemec, 2006b) at exposure levels of 5, 20 and 80 ppm were a true reflection of toxicity, and designed to examine whether more abbreviated exposure regimens targeted during lactation or more sustained exposure was necessary to replicate the effect on pup body weights. Based on the results of this study, no effects were noted on pup body weights when dams were exposed to dimethyl disulphide at concentrations of 5, 20 and 80 ppm for 1 week during lactation days 5-12 (Subset I) or lactation days 13-20 (Subset II), or for 2 weeks during lactation days 5-20 (Subset III). Therefore, the body weight effects noted for the F1 pups in the concurrent two-generation study at 5, 20 and 80 ppm were not replicated in the current study when abbreviated exposure regimens were targeted.

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
other information
Study period:
2006-02-22 to 2006-04-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline followed
Principles of method if other than guideline:
The objective of this study (WIL-160126) was to determine the critical period of exposure during lactation required to replicate lower pup body weight results at 5, 20 and 80 ppm in a concurrent two-generation reproductive toxicity study in rats (WIL-160122; Nemec, 2006). Although the lower mean body weights and body weight gains noted in the two-generation reproductive toxicity study were occasionally statistically significantly different from the control group, the effects did not occur in an exposurerelated manner. Therefore, the current study (WIL-160126) was conducted to determine if the lower pup body weight effects noted in the two-generation study could be reproduced under conditions of exposure of the F0 adults during discrete periods of F1 postnatal development. As a result, the maternal animals in the current study (WIL-160126) were exposed to the test article at exposure levels of 5, 20 and 80 ppm during 1 of 3 selected exposure periods (lactation days 5-12 [Subset I], 13-20 [Subset II] or 5-20 [Subset III]).
GLP compliance:
yes
Type of method:
in vivo
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes

All dams in all 3 subsets survived to the scheduled necropsies on lactation day 21. There were no exposure-related clinical or macroscopic findings noted for the maternal animals at any exposure level in Subsets I, II and III. Lower mean maternal body weight gains with reduced food consumption were observed in the 80 ppm group in Subsets I, II and III during the first week of exposure when compared to the control group. At 20 ppm, lower mean maternal body weight gain and reduced food consumption were noted only on lactation days 5-7 in Subset III; however, the body weight effect at this exposure level was not observed in Subset I or Subset II. Mean maternal body weights, body weight gains and food consumption in the 5 ppm group were unaffected by test article exposure. There were no effects on the number of pups found dead, and there were no clinical or macroscopic findings noted for the F1 pups as a result of maternal exposure at any concentration in Subsets I, II and III. There were no effects on pup body weights when maternal animals were exposed to 5, 20 or 80 ppm dimethyl disulfide during lactation days 5-12 (Subset I), lactation days 13-20 (Subset II) or lactation days 5-20 (Subset III).

Conclusions:
This study (WIL-160126) was conducted in order to determine if the pup body weight effects noted in the concurrent two-generation study (WIL-160122; Nemec, 2006) at exposure levels of 5, 20 and 80 ppm were a true reflection of toxicity, and was designed to examine whether more abbreviated exposure regimens targeted during lactation or more sustained exposure was necessary to replicate the effect on pup body weights. Based on the results of this study, no effects were noted on pup body weights when dams were exposed to dimethyl disulfide at concentrations of 5, 20 and 80 ppm for 1 week during lactation days 5-12 (Subset I) or lactation days 13-20 (Subset II), or for 2 weeks during lactation days 5-20 (Subset III). Therefore, the body weight effects noted for the F1 pups in the concurrent two-generation study (WIL-160122) at 5, 20 and 80 ppm were not replicated in the current study (WIL-160126) when abbreviated exposure regimens were targeted.

Justification for classification or non-classification

Based on the available data, dimethyl disulphide is not classified for reproductive and developmental toxicity according to Regulation (EC) No 1272-2008.


 


Justification of the non-classification (RAC) :



  • Effects on fertility and sexual function
    Taking into account that no treatment-related, adverse effects on sexual function and fertility were observed in relevant and acceptable studies, RAC is of the opinion that DMDS does not warrant classification for effects on fertility and sexual function.


 



  • Effects on development
    Since in five animals studies on rats and rabbits no adverse effects on viability, frequency of malformations or physical development of offspring were observed, RAC is of the opinion that DMDS does not warrant classification for effects on development. Slight reductions in body weight or body weight gains of pups or delayed ossification at the top doses were considered to be related to moderate maternal toxicity consisting of reduced food consumption and reduced maternal body weight. These effects as such are considered to be of low or minimal toxicological significance not warranting classification for developmental toxicity.


 



  • Adverse effects on or via lactation
    The existing data on DMDS do not meet not meet the following classification criteria for effects on or via lactation: (a) human evidence indicating a hazard to babies during the lactation period; and/or (b) results of one- or two-generation studies in animals which provide clear evidence of adverse effect in the offspring due to transfer in the milk or adverse effect on the quality of the milk; and/or (c) absorption, metabolism, distribution and excretion studies that indicate the likelihood that the substance is present in potentially toxic levels in breast milk. Therefore RAC is of the opinion, that DMDS does not warrant classification for adverse effects on or via lactation.

Additional information