4,4'-sulphonyldiphenol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 2013 - May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-sulphonyldiphenol

EC Number:

201-250-5

EC Name:

4,4'-sulphonyldiphenol

Cas Number:

80-09-1

Molecular formula:

C12H10O4S

IUPAC Name:

4,4'-sulfonyldiphenol

Test material form:

other: solid/white

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Weight at study initiation: see results
- Housing: 5 animals per cage in polysulfonate (H-Temp) cages, floor area about 2065 cm2 (610×435×215 mm); TECNIPLAST, Hohenpeissenberg, Germany; Polycarbonate cages (floor area about 800 cm2) to measure the motor activity; TECNIPLAST, Hohenpeissenberg, Germany. Dust-free wooden bedding.
- Diet (e.g. ad libitum): ground Kliba mouse/rat maintenance diet “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water (e.g. ad libitum): yes
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70, 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13.08.2014 To: 22.11.2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1 % suspension in drinking water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Preparation frequency: test-substance preparations were prepared at least weekly.
Storage conditions: refrigerator
Route of administration: daily gavage using 3 or 5 mL syringes
Volume to be administered: 10 mL/kg body weight

VEHICLE
- Concentration in vehicle: 0, 1, 3, 10 g/100ml
- Amount of vehicle (if gavage): 10 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses confirmed (1) the stability of the test-substance preparations over a period of 7 days in the refrigerator, (2) the homogenous distribution of the test substance in the vehicle, and (3) the correctness of the prepared concentrations.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Remarks:

for males from day 70

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

4,4´-sulphonyldiphenol was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 100 (test group 1), 300 (test group 2) and 1000 mg/kg bw/d (test group 3) over a period of 3 months. Due to severely impaired body weight development in male animals of test group 3 (1000 mg/kg bw/d), i.e. -20% on study day 63, the male animals were treated at a dose level of 600 mg/kg bw/d from study day 70 onwards. Female animals were continuously treated at limit dose.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: twice daily from monday to friday, once daily on saturdays and sundays

CLINICAL SIGNS:
- Time schedule: daily before and 2-5 h after test substance administration. Detailed clinical examinations in an open field prior to the start of the administration period and weekly thereafter.

DETAILED CLINICAL OBSERVATIONS:
- outside of cages once before the beginning of the administration period (day 0) and subsequently once a week (in the morning).

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period, during the administration period the body weight on day 0 and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly over a period of 4 days

WATER CONSUMPTION AND COMPOUND INTAKE: Yes, daily by visual inspection

OPHTHALMOSCOPIC EXAMINATION: Yes,
- Time schedule for examinations: before beginning of administration, and at the end of the administration period.
- Dose groups that were examined: all animals of the control and highest dose group.

HAEMATOLOGY: Yes, towards the end of the administration period.
- Anaesthetic used for blood collection: Yes, blood samples were taken from fasted animals by puncturing the retrobulbar venous plexus under isoflurane anesthesia.
- Animals fasted: Yes
- Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, differential blood count, reticulocytes, blood smear, prothrombin time

CLINICAL CHEMISTRY: Yes, towards the end of the administration period.
- Parameters checked: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Serum gamma glutamyl transferase, Sodium, Potassium, Chloride, Inorg. Phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins, Triglycerides, Cholesterol,

URINALYSIS: Yes, towards the end of the administration period.
- Time schedule for collection of urine / metabolism cages used for collection of urine / animals fasted: Yes, on the afternoon preceding the day fixed for urinalysis, the animals were transferred individually into metabolism cages (no food or drinking water provided). Urine was sampled overnight. On the following day, the samples were examined.
- Parameters checked: Volume, Color, Turbidity, pH value, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Microscopy of sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes, functional observational battery (FOB) and measurement of motor activity (MA).
- Time schedule for examinations: in all animals once at the end of the administration period.
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
FOB starts with passive home cage observation in the rack for a short period (about 10-30 seconds) in cages with the lids closed.
Followed by removal from the home cage for open field observation in a standard arena (50 × 50 × 25 cm).
Finally sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable.
Motor activity (MA) was measured on the same day as the FOB was performed. The examinations was performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. Animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts were counted over 12 intervals for 5 minutes per interval.

Sacrifice and pathology:

Animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

GROSS PATHOLOGY and ORGAN WEIGHTS: Yes, Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Thyroid glands, Uterus with cervix.

HISTOPATHOLOGY: Yes, All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating gland, Colon, Duodenum, Epididymides, Esophagus, Extraorbital lacrimal glands, Eyes with optic nerve (modified Davidson’s solution), Femur with knee joint, Harderian glands, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (mesenteric and axillary lymph nodes), Mammary gland (male and female) , Nose (nasal cavity), Ovaries, Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina.  

Hematoxylin and Eosin staining was performed at least from all animals of the high dose group. In liver sections of all dose groups cresyl violet stain was used for better classification of basophilic foci and GSTP immunohistochemistry was used to better detect GSTP positive (eosinophilic) hepatocellular foci.

Statistics:

Statistics of clinical examinations:
Body weight, body weight change was analysed by DUNNETT’s test.
Feces, rearing, grip strength of fore- and hindlimbs, landing foot-splay test, motor activity was analysed by KRUSKAL-WALLIS and WILCOXON test.
Statistics of clinical pathology:
Clinical pathology parameters (except for urine color and urine turbidity) were analysed by KRUSKAL-WALLIS and WILCOXON test.
Statistics of pathology
Weight of the anesthetized animals and absolute and relative organ weights were analysed by KRUSKAL-WALLIS and WILCOXON test.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
No animal died prematurely.
Soft and discolored (light brown) feces were observed in all animals of the high and intermediate test groups. The discoloration was first observed on study day 15 in male and female animals of the high dose group and on study days 84 and 85 in male and female animals of the mid dose group, respectively. Soft consistency was observed towards the end of the treatment period.
Salivation after treatment from slight to moderate was observed in all male and in 9 of 10 female animals the high dose group, in 9 of 10 male and all female animals of the intermediate dose group as well as in 6 of 10 male and 4 of 10 female animals of the low dose group. This observation was temporary and short in appearance immediately after dosing.

BODY WEIGHT AND WEIGHT GAIN
Mean body weight of male animals in the high dose group (1000/ 600 [from day 70 onwards] mg/kg bw/d) was significantly lower during the entire administration period with a maximum of -20% on study day 70. In male animals of the intermediate dose group (300 mg/kg bw/d) mean body weight was also decreased up to nearly 10% on study days 84 and 91 (see table 2).
No deviations of toxicological concern in mean body weight were observed in male animals of the low dose group (100 mg/kg bw/d).
Relevant changes in mean body weight were not observed in female animals of all treatment-groups during the entire administration period.
(see table 2).
Mean body weight change values of male animals in the high dose group (1000/ 600 [from day 70 onwards] mg/kg bw/d) were significantly lower during the entire administration period with a maximum of -33% from study day 0 to 63. In male animals of the intermediate dose group (300 mg/kg bw/d) mean body weight change values were also decreased from study day 77 onwards with a maximum of -15% from study day 0 to 84 as well as study days 0 to 91.
No relevant deviations in mean body weight change were observed in male animals of low dose group (100 mg/kg bw/d).
Relevant changes in mean body weight change were also not observed in female animals of all treatment-groups during the entire administration period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption in male animals of the high dose group (1000 mg/kg bw/d) was reduced from study day 7 to 70, with a maximum of -18% on study day 63 (see table 1). After the reduction of the dose (600 mg/kg bw/d from study day 70 onwards) the food consumption was within the usual range.
Food consumption of female animals and male animals of low and intermediate dose groups (100 and 300 mg/kg bw/d) was not influenced. (see Table 1)

WATER CONSUMPTION
No changes with regard to water consumption were observed.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related findings were observed.

HAEMATOLOGY
At the end of the administration period in both sexes of the high dose group, red blood cell (RBC) counts and hemoglobin values were decreased (-7.2/-4.4% and -5.6/-9.1% in males and females, respectively). .
In high dose females hematocrit values were decreased (-6.8%).
In high dose males mean corpuscular volume (MCV) and relative reticulocyte counts were increased (+5.5/+21%, respectively).
In high dose females prothrombin time (Hepatoquick’s test, HQT) was prolonged.
Further statistically significant changes were identified to be within the range of historical controls and therefore considered to be not treatment related.

CLINICAL CHEMISTRY
At the end of the administration period, in males of the mid and high dose groups cholesterol levels were decreased (-34/-44.3%). This effect was regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002).
Alterations in triglyceride levels in males were regarded as incidental.
In males of the high dose group creatinine values were increased (11%) and total bilirubin values were decreased (-22%). Alterations in the low dose group were considered to be not toxicologically relevant.
In females of the high dose group alkaline phosphatase activities were slightly increased (35%).

URINALYSIS
No treatment-related changes were observed.

NEUROBEHAVIOUR
Apart from incidental findings no dose- and test-substance related effects were observed in the Functional Observation battery of all dose groups (home cage observations, open-field observations and sensimotor rests/reflexes).
Likewise no dose- and test-substance related deviations to the control were noted in the motor activity measurements.

ORGAN WEIGHTS (relative)
For increased or decreased relative organ weights see Table 5.
Treatment-related and dose dependent decrease in terminal body weight in male animals.
Treatment-related increase in relative liver weights in mid and high dose group in females which correlated with histopathology. In males, the increase in relative liver weights in the high dose group was not accompanied by histopathologic findings but was outside the historical control range (This study: 2.676%, range of historical controls: 2.063% - 2.39%).
Weight increases in the adrenal gland of the high dose group of both sexes and the mid dose females were likely treatment-related although a histopathological correlate was only detected in males of the high dose group.
In the spleen relative weights were increased in high dose males and mid and high dose females, which correlated histopathologically with increased extramedullary hematopoiesis and were therefore considered treatment-related.
The decrease in thymus weight was not accompanied by histopathologic findings in females and was of questionable significance.
The statistically significant increase in relative weights of brain, epididymis, heart and testis in male animals of one or more treatment groups and increase of relative heart weights in females of the high dose was likely due to the decrease in terminal body weight.
The weight changes in the kidney were most likely also related to the decreased terminal body weight in males of all treatment groups as the histopathologically observed mineralization at the medulla/cortical junction was not believed to correlate with the weight change. In females, there was no histopathological correlate to the increased kidney weights in the mid and high dose group, therefore this was considered of equivocal significance.
The increased weights of the ovaries of high dose females were assessed as treatment-related as both absolute and relative weights were outwith the historical control range (this study absolute / relative 126 mg / 0.061%, historical control range absolute / relative 80.7 - 113.8 mg / 0.036 - 0.051%). There were no correlating histopathological findings.
There were no histopathological findings in the thyroid glands of high dose male and female animals. The weight change was therefore regarded as incidental.

GROSS PATHOLOGY
The cecum was dilated in all male animals of the high dose group.
The liver was enlarged in 8 of 10 females of the high dose group.

HISTOPATHOLOGY: NON-NEOPLASTIC
High dose male and female animals showed dilation of the cecum, characterized by a greater circumference of the cecal wall as compared to controls. A minimally increased number of apoptotic bodies were observed in the mucosa of treated animals.
Increased extramedullary hematopoiesis (mainly with reticulocytes) was observed in the spleen of both male and female animals (see table for incidence).
Male animals of the high dose group showed an increased thickness of the cortex of the adrenal gland, which was attributed to both hypertrophy and hyperplasia of cortical cells. This finding was assumed to have caused the weight increase of the adrenal glands in this test group.
Male animals of all test groups showed multifocal mineralization of tubules in the junction between cortex and medulla (termed “mineralization, medulla” in the incidence tables).
The mammary gland of mid and high dose group males showed a change from the physiological lobulo-alveolar morphology to a tubulo-alveolar appearance with smaller, more basophilic epithelial lining cells.
A dose-dependent increase in incidence and severity of centrilobular hypertrophy was noted as shown in the liver of females. Furthermore, there was an increased incidence of foci of hepatocellular alteration (especially the eosinophilic type) in the high dose females, which was also confirmed by an increased incidence of GSTP positive foci in immunohistochemistry.
Focal squamous metaplasia of the uterine glandular epithelium was noted with an increased incidence in treated female animals, which might be treatment-related.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Food consumption per day [g] 

males [g]	0	100	300	1000/600
d 3-7	18.5 +/-0	17.8 +/-0.9	18.4 +/-0.9	15.1 +/-0.1
d 10-14	21.6 +/-0.1	20.6 +/-1	22.6 +/-1.5	19.4 +/-2.8
d 17-21	23.5 +/-1.6	21.8 +/-0.4	24.4 +/-1.1	21.5 +/-0.5
d 24-28	23.1 +/-1	22.7 +/-0.4	25.9 +/-1.3	19.7 +/-1.3
d 31-35	22.5 +/-0.9	23.7 +/-1	24.9 +/-1.6	20.8 +/-1.2
d 38-42	23.6 +/-2.5	27 +/-1.6	22.9 +/-0.8	19.9 +/-0.4
d 45-49	20.7 +/-0.1	21 +/-0	20.1 +/-0.1	19.6 +/-1.2
d 52-56	20.7 +/-0.3	22.6 +/-0.6	21.4 +/-1.3	19.9 +/-1.1
d 59-63	22.4 +/-0.4	23 +/-3.3	21.9 +/-2.1	18.3 +/-2.1
d 66-70	22.5 +/-0.4	22.2 +/-1.8	22.8 +/-0.4	19.2 +/-2.3
d 73-77	21.1 +/-1.1	20.2 +/-0.6	22.1 +/-1.8	24.5 +/-5.9
d 80-84	21.1 +/-0.5	22.2 +/-0.4	21.1 +/-2.1	22.7 +/-4.4
d 87-91	22.1 +/-1.1	20.9 +/-0.8	21.5 +/-0.5	22.9 +/-4.1
females [g]	0	100	300	1000
d 3-7	12.9 +/-0.2	12.8 +/-0.39	12.6 +/-0	11.3 +/-1.1
d 10-14	14.7 +/-0.4	14 +/-0.1	14.5 +/-0.7	22.5 +/-4.7
d 17-21	14.9 +/-0.2	14.4 +/-0	15.1 +/-0.1	14.9 +/-0.5
d 24-28	16.4 +/-0.2	14.9 +/-0.5	15.6 +/-0.8	16.2 +/-0.4
d 31-35	15.2 +/-0.1	14.6 +/-0.1	14.9 +/-1	15.5 +/-0.7
d 38-42	15.6 +/-0.3	14.8 +/-0.1	15 +/-0.1	15.7 +/-0.2
d 45-49	14.7 +/-0.2	13.4 +/-0.3	16.9 +/-4.6	15.1 +/-0.2
d 52-56	16.2 +/-1.3	13.5 +/-0.1	14.1 +/-0.1	22.3 +/-9.9
d 59-63	16.3 +/-0.8	14.4 +/-0.2	15.3 +/-2.4	16 +/-4
d 66-70	17 +/-1.1	15.1 +/-0	14.8 +/-0.5	17.9 +/-2.4
d 73-77	16.2 +/-1	14.9 +/-1.5	15.1 +/-0.6	18.2 +/-5.3
d 80-84	15.7 +/-1	14.4 +/-0.2	14.9 +/-0.1	16.8 +/-0.6
d 87-91	15.1 +/-0.3	13.8 +/-0.3	14.6 +/-0.1	15.4 +/-0.4

Table 2: relative organ weights [%]

100, 300, 1000 mg/kg bw/d	males	females
Adrenals	96, 109, 177**	101, 121*, 131**
Brain	100, 103, 120**	No effect
Epididymes	101, 103, 112**	-
Heart	98, 101, 109*	101, 107, 109*
Kidneys	110*, 122**, 125**	112, 113*, 118**
Liver	No effect	109, 120**, 149**
spleen	97, 94, 119**	105, 113**, 111*
thyroid	89, 104, 116*	92, 105, 121**
thymus	No effect	100, 87, 79*
testes	104, 102, 116*	-
Ovaries	-	102, 109, 130*
uterus	-	124, 185, 95

 

Table 3: absolute body weights [g]

males [mg/kg bw]	0	100	300	1000/600
d 0	158.4 +/-6.4	157.1 +/-6.6	158.1 +/-5.5	158.2 +/-5.7
d 7	203.3 +/-9.5	199.9 +/-9.4	198 +/-9.4	189.9 +/-10**
d 14	246.7 +/-12.6	242.8 +/-14.9	240.6 +/-11.9	224.6 +/-15.4**
d 21	283.3 +/-16.9	276.7 +/-21.1	274.1 +/-11.4	253.7 +/-18.4**
d 28	311.7 +/-20	304.2 +/-26.3	298.1 +/-14.8	267.6 +/-20.1**
d 35	333 +/-23.8	328.6 +/-30.4	312.2 +/-17.2	282.1 +/-22.8**
d 42	351.4 +/-27.2	343.8 +/-33.9	326 +/-19.3	294.3 +/-26.5**
d 49	359 +/-23.1	355.8 +/-38.2	336.5 +/-19.5	302 +/-31.8**
d 56	375.1 +/-28.1	367.8 +/-40.3	345.3 +/-19.4	306.2 +/-32.3**
d 63	389.4 +/-31.6	377.8 +/-40.1	354.6 +/-19.7	312.4 +/-36.4**
d 70	396.6 +/-31.6	387.5 +/-45.4	362.3 +/-22.8	317.8 +/-36.6**
d 77	406.4 +/-31	390.6 +/-43.9	369.1 +/-22.5	326.6 +/-40.5**
d 84	413.4 +/-32.3	396.8 +/-45.4	373.8 +/-19.5	332.7 +/-42.2**
d 91	417.1 +/-31.8	400.7 +/-46.4	377.3 +/-21.7	334.7 +/-41.6**
females [mg/kg bw]	0	100	300	1000
d 0	126.1 +/-6.9	127 +/-6.9	126 +/-7.5	126.7 +/-7.8
d 7	143.7 +/-8.6	147 +/-8.2	144 +/-7.3	142.7 +/-7.9
d 14	162.9 +/-11.3	163.1 +/-10.2	162.4 +/-7.8	162.9 +/-8.8
d 21	180.1 +/-14.5	177.2 +/-9.5	173.7 +/-9.3	176.9 +/-9.4
d 28	191 +/-14.5	188.3 +/-10.7	187.6 +/-11.6	189.4 +/-11.6
d 35	201.1 +/-19	199 +/-11.8	196.4 +/-10.2	197.4 +/-11.9
d 42	208.4 +/-19.1	204.7 +/-13.8	202.8 +/-12.2	205.2 +/-12
d 49	216.8 +/-19.6	209.3 +/-13.2	207.2 +/-14.2	208.7 +/-13.6
d 56	219.3 +/-18.9	214.8 +/-13.1	212.5 +/-15.5	213.5 +/-13.8
d 63	227.2 +/-20.8	222.2 +/-13.7	216.1 +/-12.6	217.4 +/-14.4
d 70	230.2 +/-21	223.8 +/-16.2	220.6 +/-14.2	218.9 +/-15
d 77	235.5 +/-22.2	228.3 +/-14.2	222.1 +/-16.4	220.8 +/-16.6
d 84	236.1 +/-20.4	230.1 +/-13.9	224.9 +/-15.8	222.9 +/-15.9
d 91	237.3 +/-23.4	231.7 +/-14.2	225 +/-14.9	222.5 +/-15.3

Table 4: Observed effects in hematological parameters

Males [mg/kg bw/day]	0	100	300	1000
Red blood cell [tera/l]	8.71 +/-0.35	8.83 +/-0.32	8.46 +/-0.26	8.08 +/-0.39**
Hemoglobin [mmol/l]	9 +/-0.2	9 +/-0.4	8.8 +/-0.3	8.6 +/-0.2**
Hematocrit [L/L]	0.427 +/-0.014	0.426 +/-0.022	0.42 +/-0.018	0.412 +/-0.014
mean corpuscular volume [fL]	49.1 +/-1	48.2 +/-1.6	49.6 +/-1.5	51 +/-1.6**
reticulocyte counts [%]	1.5 +/-0.1	1.2 +/-0.5	1.6 +/-0.2	1.9 +/-0.4*
HQT [sec]	38.2 +/-2.2	39.2 +/-2.8	39.5 +/-2.7	40.5 +/-1.8
Females [mg/kg bw/day]	0	100	300	1000
Red blood cell [tera/l]	7.89 +/-0.31	7.832 +/-0. 2	7.76 +/-0.36	7.45 +/-0.25**
Hemoglobin [mmol/l]	8.8 +/-0.3	8.6 +/-0.2	8.5 +/-0.3	8 +/-0.3**
Hematocrit [L/L]	0.408 +/-0.012	0.406 +/-0.011	0.402 +/-0.015	0.38 +/-0.013**
mean corpuscular volume [fL]	51.8 +/-1.4	52 +/-2	51.8 +/-1.1	51 +/-1.8
reticulocyte counts [%]	1.8 +/-0.5	2 +/-0.5	2.2 +/-0.6	2.3 +/-0.5
HQT [sec]	35.5 +/-2.2	35.2 +/-1.3	36.8 +/-1.5	38.2 +/-1.2

 

Table 5: Observed effects in clinical chemistry

Males [mg/kg bw/day]	0	100	300	1000
Cholesterol [mmol/l]	1.85 +/-0.29	1.65 +/-0.36	1.23 +/-0.29**	1.03 +/-0.17**
Creatinine [mmol/l]	30.53 +/-2.2	28.2 +/-1.4*	30.8 +/-2.7	34.2 +/-3.7*
total bilirubin [µmol/l]	1.62 +/-0.2	1.57 +/-0.15	1.49 +/-0.34	1.26 +/-0.36**
alkaline phosphatase activities [µkat/l]	0.68 +/-0.12	0.8 +/-0.47	0.91 +/-0.16**	0.92 +/-0.46
Females [mg/kg bw/day]	0	100	300	1000
Cholesterol [mmol/l]	1.62 +/-0.44	1.56 +/-0.26	1.3 +/-0.29	1.33 +/-0.32
Creatinine [mmol/l]	36.6 +/-5.9	35.9 +/-2.9	34.2 +/-3.8	32.1 +/-4
total bilirubin [µmol/l]	2.52 +/-0.45	2.18 +/-0.56	2.02 +/-0.39	2.32 +/-0.41
alkaline phosphatase activities [µkat/l]	0.66 +/-0.19	0.55 +/-0.09	0.69 +/-0.25	1.01 +/-0.37*

 * p<=0.05, ** p <=0.01

Table 6:  Histopath incedence

0, 100, 300, 1000(600) mg/kg	males	females
Cecum dilation	0, 0, 0, 10	0, 0, 1, 10
spleen	0, 0, 0, 8	2, 1, 4, 10
Mammary gland	0, 0, 0, 0	0, 0, 7, 10
Liver, hypertrophy	0, 0, 0, 0	0, 2, 5, 10
Liver, foci	0, 0, 0, 0	1, 1, 1, 6
Uterus	-	0, 2, 2, 5

Applicant's summary and conclusion

Conclusions:

DHDPS was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0, 100, 300 and 1000 mg/kg bw/d over a period of 3 months. Due to severely impaired body weight development in male animals of the high dose group, i.e. -20% on study day 63, the male animals were treated at a dose level of 600 mg/kg bw/d from study day 70 onwards.

Test substance related adverse signs of systemic toxicity were observed at a dose level of 300 mg/kg bw/d and above in male animals and at a dose level of 1000 mg/kg bw/d in female Wistar rats. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 100 mg/kg bw/d in male and 300 mg/kg bw/d in female Wistar rats.

Executive summary:

Soft and discolored (light brown) feces were observed in all animals of mid and high dose groups starting on different days. These findings were assessed as being treatment-related.

Salivation after treatment was observed in most mid and high dose animals as well as in 6 of 10 male and 4 of 10 female animals of the low dose. From the temporary, short appearance immediately after dosing (or shortly before) it was concluded that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract. The effect was assessed to be non-adverse.

Cecum dilation was macroscopically identified in all high dose males. Histopathological cecum effects were noted in all male and female animals of the high dose group and in one mid dose female animal. Cecum dilation may have had a significant contribution to the decreased bw observed in the high dose males. Though the human relevance of these observations is questionable due to significant anatomical and functional differences of the cecum to rodents in which this structure is large and has a significant function in the digestion.

Regenerative anemia was identified in high dose animals (male and female) characterized by increased extramedullary hematopoiesis in the spleen correlated by findings in clinical pathology (decreased red blood cell counts and hemoglobin values, in females decreased hematocrit values, higher relative reticulocyte counts at least in males, low total bilirubin levels in males).

A weight increase of the adrenal glands of high dose male animals, which correlated with hypertrophy/hyperplasia in the cortex was regarded as adverse and treatment related.  

In absence of degenerative findings, multifocal mineralization of kidney tubules noted in males of all treatment groups was regarded as nonadverse.

In the mammary gland of male animals of the mid and high dose animals, a change from the physiological lobulo - alveolar morphology to a tubulo-alveolar appearance with smaller more basophilic epithelial lining cells (atrophy) was seen. In the literature (Rudmann et al., 2012; see also OECD, Series on Testing and Assessment, No. 106, 2009, Part4: Mammary gland [http://www.oecd.org/chemicalsafety/testing/43754898.pdf])), the occurrence of atrophy has been correlated to altered hormone levels (increased prolactin or decreased androgen) or with emaciation in male animals. This finding was regarded as adverse.

 

Increased liver weights in females of the mid and high dose was correlated with a dose-dependent centrilobular hypertrophy was seen in 2/10, 5/10, and 10/10 animals in low, mid and high dose group, respectively. Gradings were minimal in low to moderate in high dose animals correlating with the macroscopic finding “enlarged” in the high dose. In the livers of the high dose females, there was also an increased incidence of mainly eosinophilic foci of hepatocellular alteration, which were confirmed by GSTP immunohistochemistry, and considered to be adverse. Only the hypertrophy in the liver in combination with the increased liver weight of high dose females was assessed as adverse due to the presence of these foci. The hypertrophy alone in low and mid dose groups was assessed as non-adverse as no concurrent findings in clinical pathology were noted.

The increased relative liver weight in high dose males was assessed as adverse together with clinical pathology findings (lower cholesterol and higher triglyceride levels).

In the uterus, focal squamous cell metaplasia of glandular epithelium was observed in 2 females of low and mid dose group and in 5 of 10 females of the high dose group. In the high dose group this finding was assessed as possibly treatment-related and adverse due to the higher incidence. For low and mid dose group animals the finding was assessed as non-adverse as it can occur in single animals in control groups.

The increased weights of the ovaries of test group 3 females were assessed as treatment related although there were no correlating histopathological findings.