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EC number: 204-783-1 | CAS number: 126-33-0
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.
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.
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.
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 188.8.131.52.2, 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.
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.
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
24 Sept 2014
Table 2: Summary of Body Weight Changes During Gestation (g)
** = 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)
Initial body weight
Terminal body weight
Gravid uterine weight
Net body weight
Net body weight change
* = Significantly different from the control group at 0.05 using Dunnett's test
Table 4: Summary of Food consumption During Gestation (g/animal/day)
Table 5: Summary of Food Consumption During Gestation (g/kg/day)
Table 6: Summary of Fetal Data at Scheduled Necropsy
Post implantation loss
Pre implantation loss
Fetal weight (g)
No. of gravid females
0 0.0 0.00 0.00
1 0.0 0.21 0.04
* 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)
Exencephaly without open eyelid
Body shorter than normal
HC = historical control
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.
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.
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.
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