Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Additional testing proposed
An extended one-generation reproductive toxicity study in rats by the oral route is proposed.

Reason: To characterize the reproductive toxicity potential of sulfolane for classification purposes.

Justification: Reproductive/developmental effects occurred in a screening assessment study (TG 421). Under Annex IX/X, further testing for fertility and/or developmental toxicity must be considered if the criteria for classification as Repr Cat 1A or 1B are met, and the available data are adequate to support a robust risk assessment. In the screening assessment study on sulfolane, although NOAELs for reproductive and developmental toxicity have been determined, there are some significant questions about the reliability of the data. Therefore, a more definitive study is required to adequately determine the reproductive toxicity potential and thus classification of sulfolane.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental 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:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
not specified
Principles of method if other than guideline:
No further information available.
GLP compliance:
yes
Remarks:
, but not stated in report.
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River Laboratories, Japan
- Age at study initiation: 10 weeks
- Weight at study initiation: Males 355-379 g, females 209-228 g
- Housing: Individually in stainless steel cages. Each dam was moved to a plastic cage with a mat 18 days after pregnancy.
- Diet: CRF-1 (Oriental Yeast Co., LTD) ad libitum
- Water: Tap water ad libitum.
- Acclimation period: Approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 20-26°C
- Humidity: 40-70%
- Air changes: 12 times per hour.
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: Not reported.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTION
The test material was diluted with injection water to prepare a test solution. A dose was indicated by the weight of the test compound made available (after purity adjustment). The test solution was stored at room temperature under light-shielded conditions and used within 7 days after preparation, since the solution had been confirmed to cause no problems in stability when stored for 7 days under these conditions. The test compound content in the test solution of each concentration used on the starting day and on the final day of dosing of male rats was confirmed to indicate that there was no problem with the test compound concentration. The sample solution was adjusted so that the amount of 5 mL/kg body weight may be administered.
Details on mating procedure:
- M/F ratio per cage: 1 male and 1 female
- Length of cohabitation: until evidence of mating seen or for 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged Individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No information given in report.
Duration of treatment / exposure:
Two weeks prior to mating, during mating, and continuing through day 19 of gestation. The dams were then allowed to deliver their litters, which were retained until lactation day 4.
Frequency of treatment:
Daily
Details on study schedule:
No information is available.
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
700 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
The dose selection was based on the findings of the 28-day oral toxicity study, in which decreased body weight and food consumption was seen in the 700 mg/kg-day group.
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice/day

BODY WEIGHT: Yes
- Time schedule: Females day treatment initiated, twice/week until copulation confirmed, gestation days 0, 7, 14 and 21, lactation days 0 and 4. Females without evidence of mating were weighed weekly. All weighed at termination. Males: twice/week

FOOD CONSUMPTION: Yes
- Time schedule: Females twice/weekly during pre-mating period, gestation days 2, 9, 16, and 21and on lactation day 4.
Oestrous cyclicity (parental animals):
Once a day during pre-mating period until the day of copulation confirmed. Estrus occurring on two consecutive days was counted as one cycle.
Sperm parameters (parental animals):
Not determined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined as soon as possible after parturition and twice / day thereafter: number of live and dead pups and sex of pups, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Maternal animals: Mated animals on day 5 post partum, animals when evidence of mating was detected but failed to deliver 25 days after evidence of mating, animals with no evidence of mating after completion of mating period. Male animals: At the end of 49 days of treatment.

GROSS NECROPSY
- Number of implantation sites and corpora lutea were assessed.

ORGAN WEIGHTS
- Maternal animals: ovaries. Male animals: testes and epididymis.

MICROSCOPIC EXAMINATION: Yes
- Maternal animals: ovaries. Male animals: testes and epididymis. Only the control and 700 mg/kg group organs were examined.
Postmortem examinations (offspring):
External macroscopic postmortem examinations performed on all F1 pups found dead during lactation and at termination on lactation day 4. No microscopic examination of pups.
Statistics:
A homoscedasticity test according to the Bartlett method was carried out for the statistical analysis. When homoscedastic, a dispersion analysis was carried out using the one-way layout method. If the results were significant, the Dunnett method was applied. If not deemed to be homoscedastic, the one-way layout analysis utilizing the order (Kruskal-Wallis test) was carried out. If the results were found significant, the Dunnett-type test utilizing the order was applied. The copulation index, fertility index, and birth index were identified by the chi-square test.
Reproductive indices:
Gonadal function, mating behavior, implantation, and general fertility were evaluated.
Offspring viability indices:
Litter size, pup survival, sex, body weight, and the presence of gross external malformations were assessed in the offspring.
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Males: Soiled fur, diarrhea, and soft stool were observed in the 700 mg/kg group. One male rat died in the 700 mg/kg group on day 5 of administration.
Females: Soiled fur and hypersalivation were observed in the 700 mg/kg group. One female rat died in the 700 mg/kg group on lactactional day 2.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males: Significantly reduced body weights on days 4 until study termination in the 700 mg/kg group compared to controls. In the 200 mg/kg group, body weight was significantly decreased on day 4, but were similar to controls thereafter. Significantly lower food consumption was seen on days 3 and 6 in the 700 mg/kg group, but not thereafter. In the 200 mg/kg group, there was a significant on day 3 only.
Females: Significantly lower body weights in the 700 mg/kg group on days 4-11 of the pre-mating period. No significant differences in body weights between treated and control dams during the gestation and lactation periods. Significantly lower food consumption in the 700 mg/kg group on days 3 and 6 of the pre-mating period. No significant differences between treated and control dams during the pregnancy periods; there was, however, reduced in the 700 mg/kg dams on lactation day 4 compared to the controls.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
High-dose females exhibited significantly reduced number of estrous cycles during pre-mating period compared to controls (2.2 + 0.9 and 3.3 + 0.5, respectively). Four female rats were reported to exhibit irregular estrous cycles.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Relative (to body weight) ovarian weights were significantly higher in the 700 mg/kg dams.
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: complete litter loss in four dams during lactation period at 700 mg/kg/bw/day.
Key result
Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
Critical effects observed:
not specified
Clinical signs:
not specified
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not specified
VIABILITY (OFFSPRING)
The birth index (# of live pups born/# of implantation scars x 100) was significantly decreased in the 200 and 700 mg/kg dose groups compared to controls. The number of dead pups on lactation day 0, the live birth index, the number of live pups on lactation day 4, and the viability index were significantly reduced in the 700 mg/kg dose groups. Four dams in the 700 mg/kg dose group lost all of their newborn pups during the lactation period.

There was also a statistically significant decrease in delivery index (# of live pups born/# of implantation scars x 100) in the 200 mg/kg dose group.

BODY WEIGHT (OFFSPRING)
At 700 mg/kg, body weights were significantly reduced in both male and female pups on both lactation days 0 and 4. There was also a significantly significant decrease in male pup weights in the 60 mg/kg group on lactation day 0; this was considered to be spurious and not treatment-related.
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Generation:
F1
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced birth index at 200 mg/kg/bw/day
Critical effects observed:
not specified
Reproductive effects observed:
not specified

Provide table on delivery index, birth index, dead pups on LD 0, live birth index, live pups on LD 4. Add a second table with pup body weights on LD 0 and 4.

Conclusions:
Under conditions of this test, sulfolane did not influence fertility or reproductive performance. Sulfolane did, however, affect pre- and postnatal development at 200 and 700 mg/kg/day.
Executive summary:

Crj: CD(SD) rats were dosed by oral gavage with 0, 60, 200 and 700 mg/kg sulfolane. Males were dosed for 49 days (14 days pre-mating, mating and post mating). Females were dosed for 14 days pre-mating, mating, gestation, and until day 4 after delivery. In the 700 mg/kg group, body weights were significantly lower in males on days 4-49 and in females on days 4-11 (pre-mating period). There were no significant differences in body weights between treated and control dams during the gestation and lactation periods. Food consumption was not generally affected in males and females, although food consumption was significantly reduced in the high-dose females on lactation day 4. 

There were no treatment-related effects on fertility or parameters of reproductive performance. High-dose females, however, exhibited significantly reduced number of estrous cycles during pre-mating period compared to controls. At study termination, relative (to body weight) ovarian weights were significantly higher in the 700 mg/kg dams. The birth index was significantly decreased in the 200 and 700 mg/kg dose groups compared to controls. The number of dead pups on lactation day 0, the live birth index, the number of live pups on lactation day 4, and the viability index were significantly reduced in the 700 mg/kg dose groups. Four dams in the 700 mg/kg dose group lost all of their newborn pups during the lactation period. There was also a statistically significant decrease in delivery index in the 200 mg/kg dose group. At 700 mg/kg, body weights were significantly reduced in both male and female pups on both lactation days 0 and 4. There was also a significantly significant decrease in male pup weights in the 60 mg/kg group on lactation day 0; this was considered to be spurious and not treatment-related. The NOAEL for parental toxicity was considered to be 200 mg/kg-day. The NOAELs for reproductive and developmental toxicity were 200 and 60 mg/kg-day, respectively.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A reproductive/developmental screening study (TG 421) was conducted on sulfolane at doses of 0, 60, 200 and 700 mg/kg/day. The findings from this study were reviewed by Dr. Linda Roberts, a reproductive and developmental toxicology expert (ref). Dr. Roberts concludes that reproductive toxicity occurred at the middle and high dose groups of 200 and 700 mg/kg/day, respectively. Maternal toxicity, at 700 mg/kg/day, does not appear to have occurred during gestation and thus would be unlikely to have induced adverse reproductive or developmental effects. Several findings from this study are discussed below.

Fewer estrous cycles occurred in the female rats at 700 mg/kg/day during the pre-mating period. It is Dr. Robert’s opinion that this finding is questionable relative to specific reproductive toxicity for the following reasons: Stages of estrus were only evaluated during the first 14 days of the study – the premating period – and subsequently during the mating period until breeding occurred. The first week of the study is also the period during which female rats exhibited body weight loss and significantly reduced food consumption. The degree of reduced feed intake, approximately two-thirds of control values, is on the cusp of the severity found to affect fertility in rats. It would not be surprising for fertility to decline in situations of near starvation. Another reason to question the specificity of this finding is that estrous stages were measured during the first two weeks of dosing. Even 17β-estradiol may take several weeks to impair fertility. The report indicates that four females in the 700 mg/kg/day group exhibited irregular estrous cycles, but does not indicate the systemic effects [food consumption, body weight changes, general condition] these females exhibited.

Reduced birth index [no. live pups born/no. implantation scars] occurred at 200 and 700 mg/kg/day. These effects occurred in the absence of effects upon maternal body weight or food consumption during gestation, and therefore it is unlikely to be secondary to systemic toxicity as measured in this screening study. Dr. Roberts noted that, unlike the high-dose group, in which both a reduction in the number of pups born alive was noted relative to the number of implantation scars and to the number of total pups born, all pups born in the mid-dose group were primarily born alive. The statistical finding for a reduction in birth index at 200 mg/kg/day could be the result of an unfortunate reduction in pups born.

Historical control data from 38 OECD 421 studies conducted in Japan indicate that the range for control values for birth Index, 79.3% to 98.1%, brackets the results observed in the OCED 421 study conducted with Sulfolane at its mid-dose level, 90.5% (ref). This compilation of historical control data reinforces what is understood about a reproductive toxicity screen vs. a definitive testing protocol; i.e., that smaller group sizes increase variability, and that studies with lowered control reproduction values tend to have lowered values across exposure groups also (ref).

It is impossible, however, to discount the findings in the mid-dose group as the result of biological variation for both reasons of significant difference from its concurrent control and the presence of the same finding, reduced birth index, at the higher exposure level. It is a shortcoming of the screening study design that it does not have the sample size or statistical power to definitively address dose-response relationships. This is acknowledged in Annex I of the REACH legislation section 3.7.2.5.2, that the quality of evidence of results obtained from screening reproductive/developmental screening tests (e.g., OECD TG 421 and TG 422) are less reliable than that obtained through full studies.

In order to address the reliability of the reproductive effects seen in the TG 421 study on sulfolane, an extended one-generation reproductive toxicity study (TG 443) is proposed, as required under Annex IX/X.



Effects on developmental toxicity

Description of key information

In a developmental toxicity study (WIL, 2015) conducted in accordance with an appropriate guideline and in compliance with GLP, oral gavage doses of 0, 100, 200 or 500 mg/kg/day resulted in increased incidences of external fetal malformations, higher mean litter proportion of postimplantation loss and corresponding lower mean litter proportion of viable fetuses, a lower percentage of female fetuses and lower female fetus weights. The developmental no-adverse-effect-level (NOAEL) was therefore considered to be 200 mg/kg/day. Based on lower mean body weights and mean body weight changes at 200 and 500 mg/kg/day, the maternal NOAEL was considered to be 100 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 September to 19 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Sprague-Dawley (Crl:CD(SD))
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., Quebec, Canada
- Age at study initiation: approximately 87 days
- Weight at study initiation: 222 to 282g
- Fasting period before study: no
- Housing: solid bottom cages with bedding material, 2 or 3 per cage pre-mating, individually after mating
- Diet (ad libitum): PMI Nutrition International LLC Certified Rodent LabDiet 5002
- Water (ad libitum): reverse osmosis-purified on-site drinking water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 to 21.9
- Humidity (%): 38.4 to 57
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2 September 2014 To: 3 October 2014
Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was placed in a water bath (approximately 28°C) and allowed to melt prior to use. The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Concentration in vehicle: 0, 10, 20, 50 mg/mL
- Amount of vehicle: 10mL/kg
- Lot/batch no.: not stated
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Solubility of the test substance in the vehicle was established in a previous study; therefore, homogeneity assessments were not required. In addition, stability of the test substance in the vehicle at concentrations of 10 and 70 mg/mL was established following 5 and 11 days of room temperature storage.

Samples for concentration analysis were collected from the middle stratum of the first and last preparation of each dosing formulation (including the control group). One set of samples from each collection was subjected to the appropriate analyses. The remaining set of samples was stored at room temperature as back-up. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated gas chromatography method using flame ionization detection.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: until positive evidence of mating
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From Day 1 to Day 19 of gestation
Frequency of treatment:
Once daily
Duration of test:
21 days
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
25 or 26 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were selected based on a previously conducted oral dose range-finding study in pregnant rats which was conducted at dosage levels of 200, 400, 500, and 600 mg/kg/day. In that study, continual maternal toxicity in the 600 mg/kg/day group was evident with reduced mean food consumption, mean body weight gain (body weight loss was found after initial treatment) and lower mean body weights (10% lower than the control group at a maximum on gestation day 5). These effects continued in the 600 mg/kg/day group throughout the treatment period, suggesting continued toxicity to the dam. Maternal toxicity was also evident in the 500 mg/kg/day group with decreases in mean food consumption (up to 43% during the first few days of treatment) and decreases in maternal body weight gain and mean body weights. However, the 500 mg/kg/day group recovered during the second week of exposure and had only 10% reduction in body weight gain over the entire period of gestation. The 500 mg/kg/day dosage level was selected as the high-dosage level for the current study based upon the mild to moderate maternal toxicity observed with transient decreases in food consumption and body weight effects and the minimal (10%) effects on overall body weight gain during treatment period.

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily before and after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: daily gestational days 0 to 20

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined (g/animal/day) and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: daily gestational days 0 to 20

WATER CONSUMPTION: No
- Time schedule for examinations:

Haematology:
Blood samples for hematology evaluation were collected from all females at the scheduled necropsy. The animals were not fasted overnight prior to blood collection. Blood was collected from the jugular vein, using hand-held restraint, into tubes containing EDTA as the anticoagulant. The following parameters were evaluated:
Total leukocyte count (WBC)
Differential leukocyte count -
Percent
-Neutrophil (NEU)
-Lymphocyte (LYMPH)
-Monocyte (MONO)
-Eosinophil (EOS)
-Basophil (BASO)
-Large unstained cell (LUC)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus and ovaries. Spleen was weighed and preserved in neutral-buffered 10% formalin for possible further examination.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Where applicable, the litter was used as the experimental unit.

Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, clinical pathology parameters, gravid uterine weights, spleen weights, numbers of corpora lutea, implantation sites, viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-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 (Kruskal and Wallis, 1952) to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.
Historical control data:
Test Facility historical control data 21 April 1998 to 16 August 2013.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical Observations
Increased incidence of clear material around the mouth were noted in 6 or 9 females receiving 200 or 500 mg/kg/day approximately 1 hour after dosing on gestation days 12 to 18.

Body Weight
At 500 mg/kg/day a transient, statistically significant body weight loss was noted from gestation Days 1 to 3. Lower mean body weight gain was recorded then recorded during gestation days 3 and 4; thereafter body weight gain was comparable with Controls. Overall body mean weight gain during the gestation period was lower at 500 mg/kg/day due to the initial body weight loss/reduced gain and this was considered adverse.

In the 100 and 200 mg/kg/day groups, a statistically significant transient reduction in mean body weight gain was noted after the initial day of dosing (gestation day 1-2). Thereafter, mean body weight gains in these groups were comparable with the control group. The mean body weight and body weight gain were statistically significantly lower in the 200 mg/kg/day group than the control group and were considered adverse. The initial decrements in mean body weight gain in the 100 mg/kg/day group was considered test substance-related, but not adverse because there were no effects on mean absolute body weights during treatment period.

Food Consumption
Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 500 mg/kg/day group was generally lower compared to the control group during gestation days 1-8 and differences were generally statistically significant. Thereafter, mean food consumption was similar to the control group throughout the remainder of gestation. The initial decrements resulted in generally lower mean food consumption during the cumulative gestation days 1-3, 3-6, and 6-9 intervals, and the overall treatment period (gestation days 1-20); differences were generally statistically significant and considered test substance-related and adverse.

Mean food consumption in the 100 and 200 mg/kg/day groups was lower than the control group following the first 2 days of dosing (gestation days 1-3); differences were generally statistically significant. For the remainder of the treatment period, mean food consumption in the 100 and 200 mg/kg/day groups was similar to the control group. The initial lower mean food consumption in the 100 and 200 mg/kg/day groups was considered test substance-related, but not adverse because there was no effect on the overall treatment period, and no corresponding effects on mean absolute body weights.

Haematology
In the 200 and 500 mg/kg/day groups, statistically significantly lower percent eosinophils were noted compared to the control group. However, this value was within the WIL Research historical control data range for non-pregnant females. All other mean hematology values in the test substance-treated groups were similar to the control group; differences were slight, not statistically significant, and/or not present in a dose-related manner.

Necropsy Data
There were no test substance-related findings and spleen weight was unaffected by treatment.

Gravid Uterine Weight
Statistically significantly lower mean gravid uterine weight was noted at 500 mg/kg/day. At 200 mg/kg/day mean gravid uterine weight was higher than the control group and at 100 mg/kg/day was comparable to the control group.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Abnormalities:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Gestation Day 20 Laparohysterectomy Data
The mean litter proportion of postimplantation loss (early resorptions) in the 500 mg/kg/day group was statistically significantly higher when compared to the control group and accounted for all of the total resorptions in this group. A corresponding statistically significant lower mean number and litter proportion of viable fetuses was noted in this group when compared to the control group. The percentage of female fetuses and mean female fetal weights were also lower compared to the concurrent control group; the difference in female fetal weights being statistically significant (p<0.05). The percentage of female fetuses was lower than the WIL Research historical control data range. Based on these results, the test substance appears to exhibit a more pronounced effect on female fetuses and it is likely the lower number of female fetuses could be due to female implantations having a higher resorption rate than the male fetuses at 500 mg/kg/day. These effects on intrauterine growth and survival at 500 mg/kg/day were considered test substance-related and adverse.

Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 100 and 200 mg/kg/day.

Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.

Fetal Morphology Data
The numbers of fetuses (litters) available for morphological evaluation were 371(25), 329(22), 354(23), and 285(22) in the control, 100, 200, and 500 mg/kg/day groups, respectively. Malformations were observed in 1(1), 0(0), 1(1), and 6(6) fetuses (litters) in the respective dose groups.

The mean litter proportion of total malformations and variations in the 500 mg/kg/day group were higher than the control group due to higher mean litter proportions of external malformations and skeletal variations; the differences in skeletal and total variations were statistically significant, while the mean litter proportions of external and total malformations exceeded the maximum mean values in the WIL Research historical control data. These higher values were attributed to test substance-related external malformations (vertebral agenesis, exencephaly without open eyelid, anophthalmia, facial cleft, omphalocele, body shorter than normal, tarsal flexure, and microcephaly), and skeletal variations (7th cervical rib) noted at 500 mg/kg/day. There were no test substance-related external, visceral, or skeletal malformations or variations noted in the 100 and 200 mg/kg/day groups.
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1: Results of Concentration Analysis

 

Mean Concentration mg/mL (% of target)

Date of Preparation

Group 2
(10 mg/mL)

Group 3
(20 mg/mL)

Group 4
(50 mg/mL)

09 Sept 2014

9.96 (99.6)

19.9 (99.5)

50.1 (100)

24 Sept 2014

9.66 (96.6)

18.4 (92.2)

48.0 (96.0)

Table 2: Summary of Body Weight Changes During Gestation (g)

Group (mg/kg/day)

 

Control

100

200

500

Day

 

 

 

 

 

1-3

Mean
SD
N

13
3.1
25

8**
3.2
22

6**
4.4
23

-16**
7.7
22

3-6

Mean
SD
N

12
5.0
25

15
5.6
22

13
5.3
23

6**
11.1
22

6-9

Mean
SD
N

15
4.2
25

12
3.9
22

13
4.6
23

20**
8.4
22

9-12

Mean
SD
N

19
5.3
25

18
7.8
22

17
5.7
23

23
6.3
22

12-15

Mean
SD
N

19
5.2
25

21
8.8
22

19
5.2
23

17
5.6
22

15-20

Mean
SD
N

71
10.4
25

73
9.2
22

72
10.5
23

65
13.9
22

1-20

Mean
SD
N

148
17.9
25

146
13.6
22

141
19.9
23

115**
23.8
22

** = Significantly different from the control group at 0.01 using Dunnett's test

Mean differences calculated from individual differences

Nongravid weight(s) not included in calculation of mean

Table 3: Summary of Gravid Uterine Weights and Net Body Weight changes (g)

Group (mg/kg/day)

 

Control

100

200

500

Initial body weight

Mean
SD
N

253
13.9
25

252
14.0
22

250
13.8
23

251
12.0
22

Terminal body weight

Mean
SD
N

406
26.2
25

404
18.2
22

398
20.9
23

373**
27.6
22

Gravid uterine weight

Mean
SD
N

85.8
12.55
25

86.2
12.73
22

91.7
12.19
23

75.2**
20.67
22

Net body weight

Mean
SD
N

320.6
19.43
25

317.9
15.48
22

306.7*
17.09
23

297.6**
13.92
22

Net body weight change

Mean
SD
N

68
12.86
25

65.8
13.00
22

56.5**
15.62
23

47.0**
9.57
22

* = 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

Table 4: Summary of Food consumption During Gestation (g/animal/day)

Group (mg/kg/day)

 

Control

100

200

500

Day

 

 

 

 

 

1-3

Mean
SD
N

19
2.1
25

17**
1.6
22

16**
1.8
23

8**
3.0
22

3-6

Mean
SD
N

20
2.0
25

19
1.5
22

19
1.8
23

12**
3.8
22

6-9

Mean
SD
N

21
2.2
25

20
1.8
22

20
2.0
23

19**
2.1
22

9-12

Mean
SD
N

23
1.8
25

22
1.4
22

22
1.9
23

22
1.3
22

12-15

Mean
SD
N

23
1.8
25

23
2.0
22

23
2.3
23

23
2.3
22

15-20

Mean
SD
N

25
2.0
25

25
1.9
22

25
2.3
23

25
2.4
22

1-20

Mean
SD
N

22
1.6
25

21
1.3
22

21
1.6
23

20**
1.7
22

** = Significantly different from the control group at 0.01 using Dunnett's test

Nongravid weight(s) not included in calculation of mean

Table 5: Summary of Food Consumption During Gestation (g/kg/day)

Group (mg/kg/day)

 

Control

100

200

500

Day

 

 

 

 

 

1-3

Mean
SD
N

72
7.1
25

64**
4.4
22

61**
6.7
23

31**
11.2
22

3-6

Mean
SD
N

73
5.2
25

70
4.2
22

70
6.3
23

49**
13.8
22

6-9

Mean
SD
N

74
5.9
25

70
5.0
22

72
6.6
23

75
7.6
22

9-12

Mean
SD
N

74
4.2
25

72
3.6
22

73
6.2
23

80**
4.9
22

12-15

Mean
SD
N

71
6.0
25

71
5.6
22

73
6.5
23

77**
7.5
22

15-20

Mean
SD
N

68
4.6
25

68
4.2
22

69
4.9
23

75**
5.6
22

1-20

Mean
SD
N

71
4.1
25

69
3.2
22

70
4.4
23

68
4.6
22

** = Significantly different from the control group at 0.01 using Dunnett's test

Nongravid weight(s) not included in calculation of mean

Table 6: Summary of Fetal Data at Scheduled Necropsy

Dose
mg/kg/day

 

sex

Viable fetuses

Dead fetuses

Resorptions

Post implantation loss

Implantation sites

Corpora lutea

Pre implantation loss

Fetal weight (g)

No. of gravid females

M

F

early

late

0
(control)

Total
mean
SD
SE

198
7.9
1.98
0.40

173
6.9
2.10
0.42

371
14.8
2.29
0.46

 0
 0.0
 0.00
 0.00

14
0.6
0.65
0.13

 0
 0.0
 0.00
 0.00

14
0.6
0.65
0.13

385
15.4
2.38
0.48

422
16.9
2.09
0.42

37
1.5
2.26
0.45

NA
3.7
0.17
0.03

25

100

Total
mean
SD
SE

165
7.5
2.35
0.50

164
7.5
2.24
0.48

329
15.0
2.50
0.53

 0
 0.0
 0.00
 0.00

30
1.4
2.08
0.44

 0
 0.0
 0.00
 0.00

30
1.4
2.08
0.44

359
16.3
2.01
0.43

384
17.5
1.90
0.40

25
1.1
1.73
0.37

NA
3.8
0.22
0.05

22

200

Total
mean
SD
SE

174
7.6
2.11
0.44

180
7.8
2.12
0.44

354
15.4
2.02
0.42

 0
 0.0
 0.00
 0.00

14
0.6
0.78
0.16

 1
 0.0
 0.21
 0.04

15
0.7
0.88
0.18

369
16.0
1.82
0.38

404
17.6
2.90
0.61

35
1.5
3.29
0.69

NA
3.9
0.29
0.06

23

500

Total
mean
SD
SE

166
7.5
2.60
0.55

119
5.4
1.92
0.41

285
13.0*
3.58
0.76

 0
 0.0
 0.00
 0.00

36
1.6
1.47
0.31

 0
 0.0
 0.00
 0.00

36
1.6
1.47
0.31

321
14.6
3.40
0.73

369
16.8
4.08
0.87

48
2.2
2.87
0.61

NA
3.7
0.47
0.10

22

* significantly different from Control group at 0.05

Table 7: Test Substance-Related External Malformations

 

Absolute no. (% per litter)

WIL HC Mean
(range; % per litter)

Dose (mg/kg/day)

Control

100

200

500

Vertebral Agenesis

0 (0.0)

0 (0.0)

0 (0.0)

1 (0.3)

0.0 (0.0-0.4)

Exencephaly without open eyelid

0 (0.0)

0 (0.0)

0 (0.0)

1 (0.3)

0.0 (0.0-0.3)

Right anophthalmia

0 (0.0)

0 (0.0)

0 (0.0)

2 (0.6)

0.0 (0.0-0.6)

Facial cleft

0 (0.0)

0 (0.0)

0 (0.0)

1 (0.3)

0.0 (0.0-0.3)

Omphalocele

0 (0.0)

0 (0.0)

0 (0.0)

2 (0.6)

0.0 (0.0-0.3)

Body shorter than normal

0 (0.0)

0 (0.0)

0 (0.0)

1 (0.3)

0.0 (0.0-0.0)

Tarsal flexure

0 (0.0)

0 (0.0)

0 (0.0)

2 (4.9)

0.0 (0.0-0.3)

Microcephaly

0 (0.0)

0 (0.0)

0 (0.0)

1 (4.5)

0.0 (0.0-0.0)

HC = historical control

Conclusions:
Administration of tetrahydrothiophene 1,1-dioxide to pregnant Sprague Dawley (Crl:CD(SD)) rats at 0, 100, 200 or 500 mg/kg/day resulted in lower mean body weights, mean body weight changes, and food consumption at 500 mg/kg/day; lower body weights and decreased body weight change were also noted in the 200 mg/kg/day group. These changes at 200 and 500 mg/kg/day were considered to be adverse.

In addition at 500 mg/kg/day, a higher mean litter proportion of postimplantation loss (early resorptions) and corresponding lower mean litter proportion of viable fetuses, a lower percentage of female fetuses and lower female fetal weights, and increased incidences of fetal external malformations were noted which were all considered to be adverse.

Therefore 200 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for embryo/fetal development and based on the effects on body weight and food consumption the maternal NOAEL was considered to be 100 mg/kg/day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a developmental toxicity study conducted by the oral route (WIL, 2015) administration of tetrahydrothiophene 1,1-dioxide to pregnant Sprague Dawley (Crl:CD(SD)) rats at 0, 100, 200 or 500 mg/kg/day on gestation days 1 to 19 resulted in lower mean body weights, mean body weight changes, and lower food consumption at 500 mg/kg/day; lower body weights and decreased body weight change were also noted in the 200 mg/kg/day group. These changes at 200 and 500 mg/kg/day were considered to be adverse. In addition at 500 mg/kg/day, a higher mean litter proportion of postimplantation loss (early resorptions) and corresponding lower mean litter proportion of viable fetuses, a lower percentage of female fetuses and lower female fetal weights, and increased incidences of fetal external malformations (vertebral agenesis, exencephaly without open eyelid, anophthalmia, facial cleft, omphalocele, body shorter than normal, tarsal flexure, and microcephaly), and a skeletal variation (7th cervical rib) were noted which were all considered to be adverse. Therefore 200 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for embryo/fetal development and based on the effects on body weight and food consumption the maternal NOAEL was considered to be 100 mg/kg/day.


Toxicity to reproduction: other studies

Additional information

Reproductive/Developmental Toxicity

In a Reproductive/Developmental Toxicity Screening Test (OECD TG 421), Crj: CD(SD) rats were dosed by oral gavage with 0, 60, 200 and 700 mg/kg sulfolane (MHW, 1999). In the 700 mg/kg group, body weights were significantly lower in males on days 4-49 and in females on days 4-11 (pre-mating period). There were no significant differences in body weights between treated and control dams during the gestation and lactation periods. Food consumption was not generally affected in males and females, although food consumption was significantly reduced in the high-dose females on lactation day 4. There were no treatment-related effects on fertility or parameters of reproductive performance. High-dose females, however, exhibited significantly reduced number of estrous cycles during pre-mating period compared to controls. At study termination, relative (to body weight) ovarian weights were significantly higher in the 700 mg/kg dams. The birth index was significantly decreased in the 200 and 700 mg/kg dose groups compared to controls. The number of dead pups on lactation day 0, the live birth index, the number of live pups on lactation day 4, and the viability index were significantly reduced in the 700 mg/kg dose groups. Four dams in the 700 mg/kg dose group lost all of their newborn pups during the lactation period. There was also a statistically significant decrease in delivery index in the 200 mg/kg dose group. At 700 mg/kg, body weights were significantly reduced in both male and female pups on both lactation days 0 and 4. There was also a significantly significant decrease in male pup weights in the 60 mg/kg group on lactation day 0; this was considered to be spurious and not treatment-related. The NOAEL for parental toxicity was considered to be 200 mg/kg-day. The NOAELs for reproductive and developmental toxicity were 200 and 60 mg/kg-day, respectively.

Justification for classification or non-classification

The available data for tetrahydrothiophene 1,1-dioxide show significant adverse effects on fetusus and evidence of maternal toxicity. However, the period of maternal toxicity (GD 1-4) does not coincide with the developmental period for either the malformations (between GD 6-9) or the period of early resorptions (from approximately GD 8-14). In addition, the maternal toxicity observed during the entire treatment period (lower body weight gain) was not of sufficient magnitude to explain the adverse developmental effects observed. Therefore tetrahydrothiophene 1,1-dioxide requires classification for reproductive toxicity as Category 1B, H360: may damage fertility or the unborn child, according to Regulation (EC) No. 1272/2008.