Dimethyl disulphide

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl disulphide

EC Number:

210-871-0

EC Name:

Dimethyl disulphide

Cas Number:

624-92-0

Molecular formula:

C2H6S2

IUPAC Name:

(methyldisulfanyl)methane

Test animals

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

Administration / exposure

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.

Doses / concentrations

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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

1

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

2

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

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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.

Effect levels (P1)

1

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

2

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

Target system / organ toxicity (P1)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

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

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

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

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

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 Weight^b	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

Applicant's summary and conclusion

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.