Bis(4-chlorophenyl) sulphone

Endpoint summary

Description of key information

In the repeated dose key studies, subchronic oral exposure to DCDPS resulted in centrilobular hepatocyte hypertrophy associated with increased liver weight in both rats and mice (NIH, 2001). Based on these changes, the NOEL was considered to be 2 mg/kg bw/day for rats and 3.5 mg/kg bw/day for mice. Adverse liver effects were observed in male mice from a dose level of 165 mg/kg bw (NOAEL 50 mg/kg bw). While treatment related nephropathy was noted in rats at higher dose levels, no adverse effect on the kidneys was observed in mice. 
This general pattern of treatment related effects derived from the subchronic key studies was confirmed by supporting information (NIH, 2001; Poon et al., 1999) and a NOEL of 1.5 mg/kg bw  was determined in rats. However, with regard to nephropathy in rats, a moderate increase in relative kidney weight was noted after subacute exposure while no relevant change was observed after chronic exposure to DCDPS. 
Due to the physico chemical properties and the resulting toxicokinetic behaviour of DCDPS, repeated dose toxicity studies for the dermal and inhalation route are not expected to result in a substantially different systemic toxicity. With respect to animal welfare additional subchronic studies for both exposure routes are therefore not required. 

Additional information

General Remark

Data of high reliability exists for the repeated dose toxicity of DCDPS on the oral route of exposure (dietary admixture). Due to the physico chemical properties and the resulting toxicokinetic behaviour of DCDPS (see 7.1), repeated dose toxicity studies for the dermal and inhalation route are not expected to result in a substantially different systemic toxicity. Summarized, (a) no higher bioavailability and (b) no different systemic distribution and/or metabolization can be expected. Route-to-route extrapolation is therefore appropriate and with respect to animal welfare, additional subchronic studies for the inhalation/dermal exposure routes are therefore not required.

Synopsis of Key and Supporting Information

In a key subchronic toxicity study in rats (NIH, 2001), DCDPS was administered via the diet to 10 animals/sex/dose at 0, 2, 6, 19, 65 or 200 mg/kg bw/day for 14 weeks. Clinical signs, body weights, food consumption, and neurobehavioural and clinical pathological endpoints were assessed. Following necropsy, selected organs were weighed and macroscopical as well as histopathological examinations were performed. Mean body weights at 19 mg/kg bw/day or greater were significantly less than those of the controls. An increased sorbitol dehydrogenase activity was noted at 200 mg/kg bw/day (+83 %). Abolute liver weights of groups exposed to 6 mg/kg bw/day or greater and absolute kidney weights of 65 and 200 mg/kg bw/day males were significantly increased. Centrilobular hepatocyte hypertrophy was observed in males at 6 mg/kg bw/day or greater and in females at 19 mg/kg bw/day or greater, and the severities were increased in 19 mg/kg bw/day males, and 65 and 200 mg/kg bw/day males and females. Dose related increases in severity of nephropathy were observed in males at 19 mg/kg bw/day or greater with high incidences noted in both treatment and control group animals. Incidences of nephropathy in females were significantly increased at 65 and 200 mg/kg bw/day. The NOEL was established at 2 mg/kg bw/day based on centrilobular hepatocyte hypertrophy observed at higher dose levels which was associated with increased liver weight. Histopathologically, vacuolization was observed in hypertrophic hepatocytes.

In a key subchronic toxicity study in mice (NIH, 2001), DCDPS was administered via the diet to 10 animals/sex/dose at 0, 3.5, 15, 50, 165 or 480 mg/kg bw/day for 14 weeks. As in the corresponding rat study mentioned above, clinical signs, body weights, food consumption, and neurobehavioural and clinical pathological endpoints were assessed. Following necropsy, selected organs were weighed and macroscopical as well as histopathological examinations were performed. Mean body weights at 50 mg/kg bw/day or greater were minimally but statistically significantly less than those of the controls. Liver weights at 50 mg/kg bw/day or greater were significantly increased. Centrilobular hypertrophy of the liver was observed in most males exposed to 15 mg/kg bw/day or greater and in all females exposed to 165 or 480 mg/kg bw/day. The severities of findings generally increased with increasing exposure concentration. The NOEL was considered to be 3.5 mg/kg bw/day based on centrilobular hepatocyte hypertrophy and increased liver weight noted at higher dose levels. At a dose level of 165 mg/kg bw a minimal adverse necrosis was observed in males. In mice, the liver was the only target organ identified.

In a supporting dietary carcinogenicity study in rats (NIH, 2001), DCDPS was administered for 2 years to 50 animals/sex/dose at 0, 0.5, 1.5 or 5.0 mg/kg bw/day for males and 0, 1.6, 5.4 and 17 mg/kg bw/day for females. Clinical signs, body weights and food consumption were recorded periodically. Complete necropsies and microscopic examinations were performed. Mean body weights of 1.5 and 5.0 mg/kg bw/day males were generally less than those of the controls during the latter part of the study, and mean body weights of 5.4 and 17 mg/kg bw/day females were less from weeks 30 and 18, respectively. There were no increases in the incidences of neoplasms in the liver or any other organ, nor were there any liver changes indicative of overt toxicity in exposed animals. Incidences of centrilobular hepatocyte hypertrophy in 5.0 mg/kg bw/day male and 5.4 and 17 mg/kg bw/day female rats were significantly increased. Similar to the subchronic study histopathology revealed a vacuolization in hypertrophic hepatocytes. The incidences of bile duct hyperplasia and centrilobular degeneration were significantly increased in 5.4 and 17 mg/kg bw/day females. The NOEL for males was set at 1.5 mg/kg bw/day based on an increased incidence of centrilobular hypertrophy of the liver at 5 mg/kg bw/day. For females, the NOEL was established at 1.6 mg/kg bw/day based on increased incidences of centrilobular hypertrophy and bile duct hyperplasia. Adverse and centrilobular degeneration was seen in females from 5.4 mg/kg bw/day. The liver was the only target organ identified in this study.

The corresponding supporting study in mice (NIH, 2001) was performed in 50 animals/sex/dose at 0, 4, 13 or 40 mg/kg bw/day for males and 0, 3, 10 or 33 mg/kg bw/day for females. Clinical signs, body weights and food consumption were recorded periodically. Complete necropsies and microscopic examinations were performed. There were no increases in the incidences of neoplasms in the liver or any other organ. Mean body weights of high dose mice were generally less than those of the controls. The incidences of centrilobular hepatocyte hypertrophy in all exposed groups of males and in 10 and 33 mg/kg bw/day females were significantly greater than those in the controls. The NOEL was considred to be <4 mg/kg bw/day for males and 3 mg/kg bw/day for females, both based on increased incidences of centrilobular hepatocyte hypertrophy at higher dose levels. An significantly increased incidence of adverse eosinophilic foci was observed in females at 33 mg/kg bw/day.

In a supporting subacute oral toxicity study (Poon et al., 1999), DCDPS was administered via the diet to 6 male rats/dose level at 0, 0.8, 8.1 or 75.6 mg/kg bw/day for 28 days. Additional control and high dose groups were treated for 1, 2 and 3 weeks in order to assess the time course of effects. Clinical signs, body weights and food consumption were recorded periodically. Clinical laboratory investigations, bronchoalveolar lavage, liver enzyme activity determinations and DCDPS tissue level determinations were conducted. No female animals were tested and no histopathological examinations were performed. Animals treated at 75.6 mg/kg bw/day showed depressed growth rate and food consumption. Increased liver to body weight ratio was present at 8.1 mg/kg bw/day and increased kidney to body weight ratio at 75.6 mg/kg bw/day. Marked increases in hepatic benzoylresorufin O-dealkylase (BROD) and pentoxyresorufin O-dealkylase (PROD) were detected starting at 0.8 mg/kg bw/day. There was a significant decrease in methoxyresorufin O-dealkylase activity at 75.6 mg/kg bw/d. Hepatic UDP-glucuronosyltransferase and glutathione S-transferase activities increased starting at 8.1 mg/kg bw/day. A marked increase in urinary excretion of ascorbic acid was apparent starting at 0.8 mg/kg bw/day. A 3 -fold increase in serum cholesterol concentration and an increase in platelet counts by 30 % were observed at 75.6 mg/kg bw/day. Levels of thiobarbituric acid-reactive substances were increased by 3 -fold in the liver at 75.6 mg/kg bw/day but were not significantly altered in the serum. Tissue DCDPS concentrations were dose dependent. At 75.6 mg/kg bw/day, most of the treatment effects were clearly present in week 1, and the severity of the effects remained at more or less the same levels thereafter. The exceptions were hepatic BROD and PROD activities, which showed a trend towards further increases with time of treatment. Under the conditions of this supporting study lacking histopathological information, the NOAEL was 8.1 mg/kg bw/day based on, inter alia, increased relative liver (97 %) and kidney weights (20 %) and increased blood cholesterol concentration (3 -fold) at 75.6 mg/kg bw/day.

Liver Effects

Relevant changes in liver weight and histopathological liver findings noted in the key studies are depicted below.

14-WEEK FEED STUDY IN RATS

 

Dose	(mg/kg bw/day)	0	2	6	19	65	200
MALE
Organ Weight
Liver	(% bw)	3.960 ± 0.081	4.029 ± 0.070	4.768 ± 0.119**	5.481 ± 0.063**	7.082 ± 0.093**	8.471 ± 0.159**
	(% change)	0	+2	+20	+38	+79	+114
Histopathology
Liver
Hypertrophy^a	(incidence)	0/10	0/10	7/10**	10/10**	10/10**	10/10**
	(severity grade)^b	-	-	1.1	2.0	2.0	2.0
FEMALE
Organ Weight
Liver	(% bw)	3.526 ± 0.042	3.719 ± 0.057	3.968 ± 0.036**	4.609 ± 0.078**	6.471 ± 0.118**	8.316 ± 0.147**
	(% change)	0	+5	+13	+31	+84	+136
Histopathology
Liver
Hypertrophy^a	(incidence)	0/10	0/10	0/10	10/10**	10/10**	10/10**
	(severity grade)^b	-	-	-	1.0	1.9	1.9
14-WEEK FEED STUDY IN MICE
Dose	(mg/kg bw/day)	0	3.5	15	50	165	480
MALE
Organ Weight
Liver	(% bw)	1.709 ± 0.031	1.855 ± 0.039	1.789 ± 0.052	1.945 ± 0.037**	2.387 ± 0.063**	2.952 ± 0.060**
	(% change)	0	+9	+5	+14	+40	+73
Histopathology
Liver
Hypertrophy^a	(incidence)	0/10	0/10	6/10**	10/10**	10/10**	10/10**
	(severity grade)^b	-	-	1.0	2.0	3.0	3.0
Necrosis	(incidence)	0/10	0/10	1/10	3/10	7/10**	8/10**
	(severity grade)	-	-	1.0	1.0	1.0	1.0
FEMALE
Organ Weight
Liver	(% bw)	1.247 ± 0.036	1.344 ± 0.036	1.335 ± 0.024	1.564 ± 0.047**	1.802 ± 0.049**	2.290 ± 0.036**
	(% change)	0	+8	+7	+25	+45	+84
Histopathology
Liver
Hypertrophy^a	(incidence)	0/10	0/10	0/10	0/10	10/10**	10/10**
	(severity grade)^b	-	-	-	-	1.0	2.0
Necrosis	(incidence)	0/10	0/10	0/10	0/10	1/10	2/10
	(severity grade)	-	-	-	-	1.0	1.5

 

Note: values are mean ± SD of 10 animals/group
* significantly different from control (p<0.05)

** significantly different from control (p<0.01)

Based on the key results (NIH, 2001) and in due consideration of information available from supporting studies (NIH, 2001; Poon et al., 1999), a consistent pattern of liver effects was identified following chronic and subchronic exposure of rats and mice to DCDPS. A distinct and dose related liver enlargement was observed, characterized by indicators of enzyme induction and discernable microscopically by centrilobular hepatocytic hypertrophy.

 

In subchronic studies, statistically significant liver enlargement was observed from 6 mg/kg bw in rats and from 15 mg/kg in mice (NIH, 2001). The same thresholds were identified for an increase in hepatocytic hypertrophy incidence, a histopathological observation which was generally mild in severity, or at the most moderate in the high dose groups of male mice (165/480 mg/kg bw). Consistently, these dose levels in male mice were the only at which minimal necrosis was identified at a statistically significant degree.

Additional histopathological observations in the chronic rat studies were: a statistically significant increased incidence of bile duct hyperplasia (minimal severity) and a centrilobular degeneration (moderate severity) in females dosed with 5.4 and 17 mg/kg bw DCDPS. Though no necrosis was identified. Furthermore a statistically significant increase in eosinophilic foci was identified in the high dose females (33 mg/kg bw) of the chronic mouse study.In supporting enzyme induction studies, these liver effects could be correlated to an induction of cytochrome P450 isoenzymes (Mathew et al., 1996; Poon et al., 1999) and the phase II enzymes UDP-glucuronosyl- and glutathione-S-transferase (Poon et al., 1999).

Notably the chronic studies revealed, that none of these observations resulted in increased mortality and/or treatment related induction of hepatocellular adenomas/carcinomas.

 

Hepatic enzyme induction is generally recognized as a non-adverse, adaptive response associated with increases in liver weight, induction of gene expression and morphological changes in hepatocytes (Maronpot et al., 2010). These effectscan be addressed to a general chemical exposure and the need for increased metabolic activity, and are not related to an inherent toxicity of the test substance. Since this is indicative for a physiological rather than a pathological response, reversibility can be anticipated due to the dynamic character and the high adaptive capacity of the liver.

Zonal, usually centrilobular, hepatocellular hypertrophy (synonymously hepatocytomegaly) is considered a hallmark ofnon-adverseenzyme induction when there is an increased protein synthesis or increased number of cytoplasmic organelles.

Secondary to this hepatic enzyme induction, adverse effects may occur when liver responses exceed adaptive changes or induced enzymes generate toxic metabolites.

Adverse histopathological correlates of liver toxicity are e.g. hepatocyte necrosis or centrilobular degeneration. Eosinophilic foci were reported to be preneoplastic correlates.

The histopathological responses should be interpreted in conjunction with clinical pathology alterations to determine when an adaptive response becomes adverse.

 

An identical, less detailed guidance for the assessment of adverse liver effect were provided by ECETOC (2002) and Andrew (2005).

 

References:

Maronpot R.R. et al. (2010), Hepatic enzyme induction: Histopathology, Toxicologic Pathology, 38: 776-795.

The ECETOC Technical Report No. 85 (2002), “Recognition of, and Differentiation between, Adverse and Non-adverse Effects in Toxicology Studies”.

Andrew D. (2005), PSD Guidance Document: Interpretation of liver enlargement in regulatory toxicity studies.

Hard G.C. and Seely C.S. (2005), Recommendations for the interpretation of renal tubule proliferative lesions occuring in rat kidneys with advanced chronic progressive nephropathy (CPN), Toxicologic Pathology, 33:641-649.

Kidney Effects

The following table provides an overview of changes noted in the kidneys.

14-WEEK FEED STUDY IN RATS (NIH, 2001)

 

Dose	(mg/kg bw/day)	0	2	6	19	65	200
MALE
Organ Weight
Kidney (right)	(% bw)	0.352 ± 0.005	0.353 ± 0.005	0.374 ± 0.006*	0.389 ± 0.007**	0.462 ± 0.007**	0.500 ± 0.005**
	(% change)	0	0	+6	+11	+31	+42
Histopathology
Kidney
Nephropathy	(incidence)	9/10	10/10	10/10	10/10	10/10	10/10
	(severity grade)	1.0	1.0	1.0	1.8	2.9	3.8
FEMALE
Histopathology
Kidney
Nephropathy	(incidence)	0/10	0/10	1/10	2/10	5/10*	10/10**
	(severity grade)	-	-	1.0	1.0	1.0	1.2

 

Note: values are mean ± SD of 10 animals/group
* significantly different from control (p<0.05)

** significantly different from control (p<0.01)

The microscopic finding of nephropathy that was confined to rats in the subchronic study (NIH, 2001) represents a species and strain specific chronic progressive nephropathy known to occur spontaneously in Fischer 344 rats (Hard GC & Seely JC, 2005). This is supported by the high incidence of lesions noted in control group males (9/10) and by the lack of nephropathy in rats of both sexes following chronic exposure to DCDPS. This chronic progressive nephropathy is without toxicological relevance for humans.

The relative kidney weight changes are considered to be without toxicological significance in the absence of corroborative findings in related clinical laboratory parameters, macroscopical lesions and/or histopathological findings relevant for humans. In this study, the kidney weight changes noted at 6 mg/kg bw/day or greater reflected both, changes associated with chronic progressive nephropathy and, at least partially, dose-dependent decreases in terminal body weights. For example, absolute kidney weights at 65 and 200 mg/kg bw/day were only increased by +18 % and +13 %, respectively, as compared to the controls.

Overall, based on the results of the key studies and taking information from supporting studies into account, the kidney is not considered to represent a target organ of repeated exposure to DCDPS.

Conclusion

The liver is identified as the target organ of DCDPS induced toxicity based on centrilobular hepatocellular hypertrophy associated with dose-dependent increases in liver weight.

Justification for classification or non-classification

Classification of repeated dose toxicity according to Directive 67/548/EEC and Regulation 1272/2008/EC should be performed based on adverse effects observed in a 90-day repeated-dose study conducted in experimental animals and seen to occur at or below the respective guidance values. For the R48 classification according to the Directive 67/548/EEC this guidance value is50 mg/kg bw/day, for the oral route of exposure. For GHS-category 1 classification this guidance value is 10 mg/kg bw/day, for category 2 classification the guidance value range is 10-100 mg/kg bw/day, for the oral route of exposure.

In both subchronic feeding studies (rat and mice) a uniform adaptive hepatic enzyme induction was observed up to these guidance values, identified by hepatocytic hypertrophy and liver weight increase. No adverse signs of liver toxicity were identified and therefore classification is not warranted.

 

For DCDPS it was unambiguously differentiated between (i) the requirements for an adequate DNEL derivation using a conservative approach and (ii) the target organ toxicity related classification according to Directive 67/548/EEC and Regulation 1272/2008/EC. Therefore, the lowest systemic NOEL of 1.5 mg/kg bw/day was applied as the point of departure for the DNEL derivations. This No Observed Effect Level was identified in a chronic feeding study in rats based on an adaptive increase in liver weights accompanied by centrilobular hepatocellular hypertrophy in males (NIH 2001). The NOEL was subsequently compared to a combined systemic exposure estimate for both, inhalation and dermal exposure. The DNELs are therefore considered to adequately protect workers (no exposure of the general population) against potential adaptive liver effects associated with DCDPS exposure.