Registration Dossier

Administrative data

Description of key information

Sprague Dawley  rats of received diets containing 100, 200 or 2000 ppm diphenyl sulphone for a period of 13 weeks, corresponding to mean test article intake values of 8, 16 and 164 mg/kg bw/day in males and 9, 19 and 206 mg/kg bw/day in females, respectively.  NOEL was 100 ppm (8-9 mg/kg bw/day) and NOAEL was considered to be 200 ppm (16-19 mg/kg bw/day). Adaptive changes were seen at  200 ppm, as demonstrated by increased liver weights and cellular hypertrophy, whereas toxicological changes were seen at 2000 ppm (corresponding with 164 and 206 mg/kg bw/day) as demonstrated by changes in body weight gain, food intake, blood and urine chemistry, liver and kidney weights, histopathology, peroxisome proliferation and electron microscopy of liver.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study is non-GLP (1981), however it has been performed according to state of the art and actual methods and is therefore considered reliable, relevant and adequate.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent
- Age at study initiation: 41 days
- Weight at study initiation: males 136-195g; females 95-159g
- Fasting period before study: not applicable
- Housing: housed in groups of 5 by sex in stainless steel grid cages over cardboard lined trays
- Diet (e.g. ad libitum): diets were available continuously for 13 weeks with the exception of 16 hour periods of deprivation associated with the collection of blood and urine samples
- Water (e.g. ad libitum): water was available continuously for 13 weeks with the exception of 16 hour periods of deprivation associated with the collection of blood and urine samples
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 40-70%
- Air changes (per hr): at least 12 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs darkness/ 12 hrs light

IN-LIFE DATES: From: 16 september 1980 To: 16-20 december 1980
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly intervals
- Mixing appropriate amounts with (Type of food): powdered diet (rat and mouse No 1, Modified, BP Nutrition Ltd., Stepfield, Witham, Essex)
- Storage temperature of food: room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable
- Concentration in vehicle: not applicable

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogenicity and stability of the test article in the powdered diet were determined prior to the study. The test article was shown to mix homogeneously in the speicfied diet and test diets were found to be stable over a 14-day period (HLE-report No. 2486-72/203).
Routine analysis of test diets for achieved concentration was performed on diets prepared prior to the start of treatment and again during weeks 4,8 and 13 of treatment. On each occasion triplicate samples of each batch of diet prepared, together with triplicate samples of the control diet provided were analysed for test article concentration.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
With the exception of certain periods of blood and urine collection animals were allowed free access to food with or without (control group) test compound.
Remarks:
Doses / Concentrations:
100 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
200 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
2000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
per dose: 30 animals (15 male and 15 female)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dietary concentrations, which were selected by the study sponsor in the light of results from a 4 week feeding study
- Rationale for animal assignment (if not random): the animals were allocated to cages and to treatment groups using the sponsor's replicate system
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of the test, at weekly intervals throughout the study and again on the day of necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No for individual data, but mean food intake for groups of 5 (cage means) and dose groups are available.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY: +-
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not calculated, only deduced.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before treatment and repeated during weeks 6 and 13 for all animals from groups 1 and 4
- Dose groups that were examined: groups 1 and 4 (control and high)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before start of treatment and again after 5 and 12 weeks of treatment
- Anaesthetic used for blood collection: Yes, diethyl ether
- Animals fasted: Yes , 16 hours
- How many animals: 40 (10 males and 10 femaels of group 1 and 4)
- Parameters checked in table were examined: yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before start of treatment and again after 5 and 12 weeks of treatment
- Animals fasted: Yes, 16 hours
- How many animals: 40 (10 males and 10 femaels of group 1 and 4)
- Parameters checked in table were examined: yes

URINALYSIS: Yes
- Time schedule for collection of urine: before start of treatment and again after 5 and 12 weeks of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table) a full macroscopic examination of all tissues and organs in situ was performed and organs were weighed prior to fixation (see table below)
HISTOPATHOLOGY: Yes (see table) Samples of the tissues and organs (mentioned below), with exception of eyes which were fixed in Davidson's fluid, were fluid in 10% neutral buffered formalin.
Other examinations:
Aminopyrine N-demethylase (APDM) activity and electron microscopy on liver samples (6 males and 6 females/group)
Statistics:
Evaluation of body weight, haematological, APDM activity and organ weight data was performed using analysis of variance followed bu Students 't' test. Group mean differences for selected biochemical parameters were evaluated using the Kruskal-Wallis test and Wilcoxon rank sum test. The significance of the incidence of selected pathological findings was assessed using the Chi squared method with Yate's correction.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment- related clinical signs. One female animal given 200 ppm diphenylsulphone died on day 94 under ether anaesthesia during blood collection. Death was considered to have resulted from ether overdosage and not from treatment.
Mortality:
no mortality observed
Description (incidence):
There were no treatment- related clinical signs. One female animal given 200 ppm diphenylsulphone died on day 94 under ether anaesthesia during blood collection. Death was considered to have resulted from ether overdosage and not from treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean overall weight gains of animals of both sexes given 2000 ppm diphenylsulphone were significantly lower at the 1% level of probability than those of the controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males given 200 or 2000 ppm diphenylsulphone showed a slight reduction in food intake over the treatment period as a whole (no effect in treated females).
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Markedly reduced in animals of both sexes given 2000 ppm but unaffected at dietary levels of 200 ppm and below.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ocular changes that could be ascribed to administration of the test article
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Minor reduction in mean red blood cell indices in animals of both sexes given 2000 ppm (at week 13) when compared to controls. All individual data were considered normal.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
An increase in the prevalence and degree of positive reactions of ketones and reducing substances was seen in the urine of both sexes given 2000 ppm diphenylsulphone.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see below
Gross pathological findings:
no effects observed
Description (incidence and severity):
no lesions that could be attributed to administration of the test article.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below
Histopathological findings: neoplastic:
no effects observed
Details on results:
ELABORATION OF SOME PARAMETERS

FOOD CONSUMPTION AND COMPOUND INTAKE (feeding study): Mean test article intake was 8, 16 and 164 mg/kg bw/day in males and 9, 19 and 206 mg/kg bw/day in females at the 100, 200 and 2000 ppm dose groups, respectively. Values ranged during the 13 week dosing period from 13 to 6, 27 tot 11 and 240 to 120 mg/kg bw/day in males and from 13 to 8, 26 to 15 and 254 to 157 mg/kg bw/day in females at the 100, 200 and 2000 ppm dose groups. Based on the growth curve and high inital food intake, test article intake was highest in the younger animals and decreased during the study.

CLINICAL CHEMISTRY: The mean plasma cholesterol levels of animals of both sexes given 2000 ppm were elevated at both the week 6 and 13 investigations, whilst an elevation of the plasma triglyceride level was seen in the same animals at the former investigation only. Mean plasma alkaline phosphatase levels were reduced in animals of both sexes after 6 weeks and in the males only, after 9 weeks of treatment at 2000 ppm diphenyl sulphone.

ORGAN WEIGHTS: Statistically significant increases in mean relative liver weight were seen in animals of both sexes receiving 200 or 2000 ppm. The mean relative kidney weights of male animals given 200 ppm and animals of both sexes given 2000 ppm were also significantly greater than control values. These effects were considered to be treatment-related. However a slight increase in female mean relative kidney weight at the 100 ppm level which also achieved statistical significance, was thought to have been spurious. The increase in relative brain weight shown by animals of both sexes given 2000 ppm was considered to be a reflection of lower terminal body weights.

HISTOPATHOLOGY: NON-NEOPLASTIC: Microscopic examination revealed hepatocellular hypertrophy in animals of both sexes given 2000 ppm and also in femals given 200 ppm. The slight hypertrophy seen in a few animals from the remaining treated groups could not be distinguished statistically from the minimal hypertrophy observed in a proportion of control animals. In addition, profound kidney changes consisting of corticotubular degeneration and regeneration were seen in males given 2000 ppm. An increase in the degree of corticotubular eosinophilic droplet formation was also demonstrated for males given 200 or 2000 ppm.

OTHER FINDINGS

LIVER APDM activity: A significant increase in mean APDM activity was apparent for group 4 rats of both sexes when compared to corresponding control values. There was no effect on APDM activity in rats from group 2 and 3.

ELECTRON MICROSCOPY: all animals receiving 2000 ppm showed a proliferation of liver smooth endoplasmic reticulum. Minimal changes were noted in 2/6 rats receiving 200 ppm diphenyl sulphone and were considered to have been a non-specific response associated with binding of xenobiotic to albumin, or competition with metabolic pathways. No electron microscopy was conducted on animals at the 100 ppm dose level. Electron microscopic examination also revealed increased lipid vacuolation in some hepatocytes from 2 males and 1 female rat given 2000 ppm diphenyl sulphone.
Dose descriptor:
NOEL
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: organ weights; histopathology changes at 200 and 2000 ppm
Dose descriptor:
NOEL
Effect level:
8 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: mean test article intake (ranges: 13 to 6, 27 mg/kg bw/day during 13 weeks)
Dose descriptor:
NOEL
Effect level:
9 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: mean test article intake (range from 13 to 8, 26 mg/kg bw/day during 13 weeks)
Dose descriptor:
NOAEL
Effect level:
200 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Changes in body weight gain, food intake, blood and urine chemistry, organ weights, histopathology, peroxisome proliferation and electron microscopy at 2000 ppm
Dose descriptor:
NOAEL
Effect level:
16 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: mean test article intake (range from 27 to 11 mg/kg bw/day during the 13 weeks)
Dose descriptor:
NOAEL
Effect level:
19 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: mean test article intake (range from 26 to 15 mg/kg bw/day during the 13 weeks)
Critical effects observed:
not specified
Conclusions:
NOEL was 100 ppm (8-9 mg/kg bw/day). Target organs were the liver and kidneys at the dose levels of 200 and 2000 ppm, as demonstrated by organ weights, histopathology and additional investigations (Aminopyrine N-demethylase and electron microscopy).
Executive summary:

Sprague Dawley CD rats of received diets containing 100, 200 or 2000 ppm diphenyl sulphone for a period of 13 weeks, corresponding to mean test article intake values of 8, 16 and 164 mg/kg bw/day in males and 9, 19 and 206 mg/kg bw/day in females at the 100, 200 and 2000 ppm dose groups, respectively. One female animal given 200 ppm diphenyl sulphone died on day 94 from ether overdosage and not from treatment. There were no treatment-related mortalities, clinical signs and gross pathological findings. The mean overall weight gains of animals of both sexes given 2000 ppm diphenyl sulphone were significantly lower than controls. Males given 200 and 2000 ppm diphenyl sulphone showed a slight reduction in food intake over the treatment period as a whole; there was no effect on food intake in treated females. Food conversion efficiency was markedly reduced in animals of both sexes given 2000 ppm but was unaffected at dietary levels of 200 ppm and below. Minor reductions in mean red blood cell indices were seen in animals of both sexes given 2000 ppm at week 13 when compared to control values, however, all individual data were considered normal. The mean plasma cholesterol levels of animals of both sexes given 2000 ppm were elevated at both the week 6 and 13 investigations, whilst an elevation of the plasma triglyceride level was seen in the same animals at 6 weeksonly. Mean plasma alkaline phosphatase levels were reduced in both sexes after 6 weeks and in the males only, after 9 weeks of treatment at 2000 ppm. An increase in the prevalence and degree of ketones and reducing substances was seen in theurine specimens of animals of both sexes given 2000 ppm. Statistically significant increases in mean relative liver weight were seen in animals of both sexes at 200 or 2000 ppm. The mean relative kidney weights of males given 200 ppm and both sexes given 2000 ppm were also significantly greater than control values. These effects were considered to be treatment-related, however a slight increase in female mean relative kidney weight at the 100 ppm level which also achieved statistical significance, was thought to have been spurious. The increase in relative brain weight shown by animals of both sexes given 2000 ppm was considered to be a reflection of lower terminal body weights. Microscopic examination revealed hepatocellular hypertrophy in animals of both sexes at 2000 ppm and also in females at 200 ppm. The slight hypertrophy seen in a few animals from the remaining treated groups could not be distinguished statistically from the minimal hypertrophy observed in a proportion of control animals. In addition, profound kidney changes consisting of corticotubular degeneration and regeneration were seen in males given 2000 ppm. An increase in the degree of corticotubular eosinophilic droplet formation was also demonstrated for males given 200 or 2000 ppm. A statistically significant increase in liver aminopyrine N-demethylase activity was seen in animals of both sexes given the highest dietary level. All animals receiving 2000 ppm showed a proliferation of liver smooth endoplasmic reticulum. Minimal changes were noted in 2/6 rats receiving 200 ppm diphenylsulphone and were considered to have been a non-specific response associated with binding of xenobiotic to albumin, or competition with metabolic pathways. Electron microscopic examination also revealed increased lipid vacuolation in some hepatocytes from 2 ma1es and one female rats given 2000 ppm diphenylsu1phone. On the basis of these findings 100 ppm dipheny1su1phone (corresponding wiht 8 -9 mg/kg bw/day in male and female rats) was considered to represent a NOEL. The dose level of 200 ppm (corresponding with 16 -19 mg/kg bw/day in male and female rats) was considered to represent a NOAEL.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
16 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
High quality

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In this registration dossier, a subchronic dietary (90 day) toxicity study with diphenyl sulphone was provided as the key study for repeated dose toxicity (Hazleton Laboratories, 1981). Based on the exposure and physicochemical properties, there is no indication for significant dermal absorption nor inhalation risk, therefore this study was considered adequate and relevant. The study also covers the 28 -day study, which was waived in this dossier.

Sprague Dawley CD rats of received diets containing 100, 200 or 2000 ppm diphenyl sulphone for a period of 13 weeks, corresponding to mean test article intake values of 8, 16 and 164 mg/kg bw/day in males and 9, 19 and 206 mg/kg bw/day in females at the 100, 200 and 2000 ppm dose groups, respectively. One female animal given 200 ppm diphenyl sulphone died on day 94 from ether overdosage and not from treatment.There were no treatment-related mortalities, clinical signs and gross pathological findings. The mean overall weight gains of animals of both sexes given 2000 ppm diphenyl sulphone were significantly lower than controls. Males given 200 and 2000 ppm diphenylsulphone showed a slight reduction in food intake over the treatment period as a whole; there was no effect on food intake in treated females. Food conversion efficiency was markedly reduced in animals of both sexes given 2000 ppm but was unaffected at dietary levels of 200 ppm and below. Minor reductions in mean red blood cell indices were seen in animals of both sexes given 2000 ppm at week 13 when compared to control values, however, all individual data were considered normal.The mean plasma cholesterol levels of animals of both sexes given 2000 ppm were elevated at both the week 6 and 13 investigations, whilst an elevation of the plasma triglyceride level was seen in the same animals at 6 weeks only. Mean plasma alkaline phosphatase levels were reduced in both sexes after 6 weeks and in the males only, after 9 weeks of treatment at 2000 ppm. An increase in the prevalence and degree of ketones and reducing substances was seen in the urine specimens of animals of both sexes given 2000 ppm.Statistically significant increases in mean relative liver weight were seen in animals of both sexes at 200 or 2000 ppm. The mean relative kidney weights of males given 200 ppm and both sexes given 2000 ppm were also significantly greater than control values. These effects were considered to be treatment-related, however a slight increase in female mean relative kidney weight at the 100 ppm level which also achieved statistical significance, was thought to have been spurious. The increase in relative brain weight shown by animals of both sexes given 2000 ppm was considered to be a reflection of lower terminal body weights. Microscopic examination revealed hepatocellular hypertrophy in animals of both sexes at 2000 ppm and also in females at 200 ppm. The slight hypertrophy seen in a few animals from the remaining treated groups could not be distinguished statistically from the minimal hypertrophy observed in a proportion of control animals. In addition, profound kidney changes consisting of corticotubular degeneration and regeneration were seen in males given 2000 ppm.An increase in the degree of corticotubular eosinophilic droplet formation was also demonstrated for males given 200 or 2000 ppm. A statistically significant increase in liver aminopyrine N-demethylase activity was seen in animals of both sexes given the highest dietary level. All animals receiving 2000 ppm showed a proliferation of liver smooth endoplasmic reticulum. Minimal changes were noted in 2/6 rats receiving 200 ppm diphenyl sulphone and were considered to have been a non-specific response associated with binding of xenobiotic to albumin, or competition with metabolic pathways.Electron microscopic examination also revealed increased lipid vacuolation in some hepatocytes from 2 males and one female rats given 2000 ppm diphenyl su1phone. On the basis of these findings 100 ppm diphenyl sulphone (corresponding with 8 -9 mg/kg bw/day in male and female rats) was considered to represent a NOEL. The dose level of 200 ppm (corresponding with 16 -19 mg/kg bw/day in male and female rats) was considered to represent a NOAEL.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Key study

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Justification for classification or non-classification

Toxicological effects were only observed at 2000 ppm dietary levels, corresponding with 164 and 206 mg/kg bw/day, therefore classification is not warranted according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC) and CLP regulation (EC No. 1272/2008 of 16 December 2008).