Bis(4-hydroxy-N-methylanilinium) sulphate

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from NTRL report

Data source

Reference

Reference Type:

secondary source

Title:

Initial Submission: Letter From Eastman Kodak Co To Usepa Regarding Toxicity Studies Of 4-(Methylamino)Phenol Sulfate Attachments And Cover Letter Dated 082892- 90-Day Toxicity Study of p-Methylaminophenol Sulfate in Sprague Dawley Rats

Author:

James R. Harr, Milan S. Vlaovic, Robert E. Emmons

Year:

1982

Bibliographic source:

NTRL, 1982, OTS0570966

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

A repeated dose oral toxicity study was performed to determine the toxic nature of Elon using male and female Crl: COBS®, CD®, SD, BR rats for 91-94 days.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(4-hydroxy-N-methylanilinium) sulphate

EC Number:

200-237-1

EC Name:

Bis(4-hydroxy-N-methylanilinium) sulphate

Cas Number:

55-55-0

Molecular formula:

C14H20N2O6S

IUPAC Name:

bis(4-hydroxy-N-methylanilinium) sulfate

Test material form:

solid: crystalline

Details on test material:

- Name of test material: p-Methylaminophenol sulfate- IUPAC name: Bis(4-hydroxy-N-methylanilinium) sulphate- Molecular formula: C14H20N2O6S- Molecular weight: 344.386 g/mole- Smiles:CNc1ccc(cc1)O.CNc1ccc(cc1)O.OS(=O)(=O)O- Inchl: 1S/2C7H9NO.H2O4S/c2*1-8-6-2-4-7(9)5-3-6;1-5(2,3)4/h2*2-5,8-9H,1H3;(H2,1,2,3,4)- Substance type: Organic- Physical state: Solid crystalline (off white - white)

Specific details on test material used for the study:

- Name of test material : Elon- Molecular formula : C7H9NO.1/2H2O4S- Molecular weight : 344.386 g/mol- Substance type: Organic- Physical state: No data- Impurities (identity and concentrations): No data

Test animals

Species:

rat

Strain:

other: Crl: COBS®, CD®, SD, BR

Details on species / strain selection:

No data available

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: No data available- Age at study initiation: No data available- Weight at study initiation: 75-100 g- Fasting period before study: No data available- Housing: Five animals per cage in suspended stainless steel wire cages in a controlled environment. - Diet (e.g. ad libitum): Ground feed (Purina Ground Rodent Chow 5001®), ad libitum- Water (e.g. ad libitum): Potable water, ad libitum- Acclimatization period: 13 daysENVIRONMENTAL CONDITIONS- Temperature (°F): 73-75˚F- Humidity (%): 40-55%- Air changes (per hr): 6% fresh air replacement during each air cycle (157 cycles/hr)- Photoperiod (hrs dark / hrs light): 12-hrs light/12-hrs dark

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

No data available

Vehicle:

water

Remarks:

Distilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Elon was dissolved in distilled water to give a dose level of 0, 0.3, 1.0 or 3.0% (equal to approx. 0, 30, 100 or 300 mg/kg bw). The solution was prepared daily by weighing appropriate amounts of the compound into volumetric flasks and adding sufficient distilled water to fill the flasks to volume. Dose solutions were used within two hours of preparation.DIET PREPARATION- Rate of preparation of diet (frequency): No data available- Mixing appropriate amounts with (Type of food): No data available- Storage temperature of food: No data availableVEHICLE- Justification for use and choice of vehicle (if other than water): Distilled water- Concentration in vehicle: 0, 0.3, 1.0 or 3.0% (equal to approx. 0, 30, 100 or 300 mg/kg bw)- Amount of vehicle (if gavage): 10 ml/kg bw/day- Lot/batch no. (if required): No data available- Purity: No data available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Biweekly aliquots of the dose solutions were analyzed by the Industrial Laboratory for the concentration of the compound. In addition, the pH of the last five aliquots was measured.

Duration of treatment / exposure:

91-94 days

Frequency of treatment:

5 days per week

Doses / concentrations

Remarks:

Doses/Concentrations: 0, 0.3, 1.0 or 3.0% (equal to 0, 30, 100 or 300 mg/kg bw)

No. of animals per sex per dose:

Total: 160 ratsControl: 20 males,20 females30 mg/Kg bw: 20 males, 20 females100 mg/Kg bw: 20 males, 20 females300 mg/Kg bw: 20 males, 20 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No data available- Rationale for animal assignment (if not random): During the 13 day quarantine period, seven (5 male, 2 female) rats developed minimal clinical abnormalities and were discarded. On day 1 of the exposure period the remaining population (95 M, 98 F) was culled to 80 males and 80 females by removing rats with outlying body weights. The test population (80 M, 80 F) was then randomly distributed by sex into four cages of five rats each, per sex per dose group. The four cages per sex per dose group (20 rats) were arranged in a vertical column on a rack, one sex per rack. The culled rats (outlying body weights) were housed on a third rack maintained in as a sentinal population the same room.On day 2 of the exposure period, six of the sentinal rats were randomly selected by sex to replace six rats in the test population that were accidentally killed during the first dosing. These rats received one dose less than the others in the test population.- Rationale for selecting satellite groups: No data available- Post-exposure recovery period in satellite groups: No data available- Section schedule rationale (if not random): No data available

Positive control:

No data available

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes- Time schedule: before and one and five hours after each dose- Cage side observations included: Mortality and morbundityDETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: Before and one and five hours after each doseBODY WEIGHT: Yes- Time schedule for examinations: On days zero, three, six, and generally weekly thereafter FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, twice weekly, whenever a rat died, and daily from day 27 through 31- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data availableFOOD EFFICIENCY: No data availableWATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data availableOPHTHALMOSCOPIC EXAMINATION: No data availableHAEMATOLOGY: Yes - Time schedule for collection of blood: On days 91 through 94- Anaesthetic used for blood collection: Carbon dioxide- Animals fasted: Yes, overnight- How many animals: First two or three rats from each cage (from 82 of the 132 surviving rats)- Parameters examined: Electrophoresis of hemoglobin, osmotic fragility and effects on erythrocytes, hemoglobin concentration, RBC, HCT, MCH, MCV, MCHC, reticulocytes, Heinz bodies, RBC fragility, WBC, lymphocytes, monocytes, eosinophils, basophils and platelets.CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: On days 91 through 94- Animals fasted: Yes, overnight- How many animals: First two or three rats from each cage (from 82 of the 132 surviving rats)- Parameters examined: AST, ALT, LDH, AP, glucose, urea nitrogen and creatinine. URINALYSIS: Yes- Time schedule for collection of urine: Day 80 from first rat of each cage- Metabolism cages used for collection of urine: No data available- Animals fasted: No data available- Parameters examined: Urines was analysed for electrolytes, inorganic and organic components-related to the compound incl. concentration of N-acetyl-β-glucosaminidase (NAG), a-Glutamyltransferase (GGT), creatinine, protein, ketone, bilirubin, nitrite, urobilinogen, sscorbic acid, pH, osmolality, volume, color, appearance and microscopic examination of sediment.NEUROBEHAVIOURAL EXAMINATION: No data availableOTHER: During days 84 through 86, systolic blood pressure and electrocardiograms were determined on the first rat in each cage except the females in the 300 mg/kg group.

Sacrifice and pathology:

GROSS PATHOLOGY: YesEach of the 160 rats was necropsied. The 28 that died or became moribund were necropsied as they were discovered. The 132 rats that survived the exposure period were killed and necropsied on days 91 through 94, one cage/sex/ dose group/day. Prior to necropsy, the rats were fasted overnight, anesthesized with carbon dioxide, bled from the abdominal vena cava and then exsanguinated. The exsanguinated cadavers were weighed.HISTOPATHOLOGY: YesThe lungs were filled in situ with 10% neutral buffered formalin, the cadaver was dissected and¨tissues were collected for histologic processing. Pathology was conducted on trachea, lungs, thymus, heart (weighed), tongue, esophagus, stomach , duodenum, jejunum, ileum, cecum, colon, liver (weighed), kidneys (weighed), urinary bladder, pituitary gland, adrenal glands (weighed), pancreas, thyroid glands, parathyroid glands, spleen (weighed), mesenteric lymph nodes, bone marrow, brain (weighed), quadriceps femoris, eyes, rib, femur, salivary glands, skin, sciatic nerve, cervical spinal cord, ovaries (weighed), Fallopian tubes, uterus, cervix uteri, vagina, mammary gland (female), testes (weighed), epididymides and accessory sex organs (males).

Other examinations:

No data available

Statistics:

Data were analyzed by Bartlett's Test, one way analysis of variance and Duncan's multiple comparison test. Statistically significant effects were those with p < 0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs included 33 sporadic and 398 compound and dose-related effects. Compound and dose related effects were green brown discoloration of urine, decreased activity and moribundity and mortality. In the 300 mg/kg group, activity was decreased during weeks five through fourteen.In the 300 mg/kg group, activity was decreased during weeks five through fourteen. Urine discoloration was green (minimal) during exposure weeks one and two and brown (severe) during weeks three through fourteen. In the 100 mg/kg group, urine was brown (moderate) during weeks five through fourteen. In the 30 mg/kg group urine was brown (slight) during weeks nine through fourteen.

Mortality:

mortality observed, treatment-related

Description (incidence):

During the exposure period three rats became moribund and 25 others died. The 28 moribund or dead rats included 11 males and 17 females; 20 were in the 300 mg/kg group, 7 in the 100 mg/kg group and 1 (female No. 689) in the 0 mg/kg group. The females died (days 13 to 65, doses 10 to 45) earlier in the exposure period than the males (days 16 to 91, doses 13 to 64). There were no significant differences in compound related effects between sexes. There was no qualitative difference in effects between those that died or became moribund and those that were necropsied at the end of the exposure period (days 91 through 94).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no statistically or biologically significant differences in the mean body weight (by sex) among the dose groups. On days 3, 6 and 13 the average body weights of the males in the 300 mg/kg group were 8.5, 6.7 and 4.3% less than in the other three dose groups. This difference was not biologically significant and did not occur in the females. The 160 rats were each weighed up to 16 times during the exposure period at 1 to 9 day intervals. There were never two successive weight losses in the same rat and only two of the 28 rats that died or became moribund lost weight immediately before death.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There were no biological or statistical differences in feed consumption among the dose groups except during days 1 to 3 in the 300 mg/kg group. Average feed consumption for days 1 through 3 (measured on day 3) was 30% less (males and females) in the 300 mg/kg group than in the 0, 30 or 100 mg/kg groups. This observation was associated with decreased weight gain in the male rats in the 300 mg/kg group on days 3 (8.57.), 6 (6.7%) and 13 (4.3%). There was no significant decrease in weight gain in the female rats in the 300 mg/kg group on days 3, 6 and 13.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg, a moderate decrease in hematocrit, a moderate decrease in hemoglobin and erythrocyte concentrations, a large increase in the incidence of reticulocytes and Heinz bodies and a slight increase in mean corpuscular hemoglobin concentration was observed. There were no other significant differences in hematologic parameters between the 300 and 0 mg/kg groups.When exposed to 100 mg/kg, a slight decrease in hematocrit, a slight decrease in hemoglobin and erythrocyte concentrations and a slight increase in the incidence of reticulocytes and Heinz bodies. There were no other significant differences in hematologic parameters between the 100 and 0 mg/kg groups.At 30 mg/kg, there were no statistically significant differences in any hematologic parameters between the 30 and 0 mg/kg groups. However, there were compound related trends in erythrocyte concentration and in the incidence of Heinz bodies.Erythrocyte Morphology: Compound related morphologic effects in erythrocytes were slight (100 mg/kg group) to moderate (300 mg/kg group) polychromasia, macrocytosis, anisocytosis, siderocytosis and incidence of Howell-Jolly bodies. In addition, minimal to minor hypochromasia, microcytosis, spherocytosis and incidence of target cells occurred in 3 of 22 rats observed in the 100 mg/kg group and in 8 of 20 observed in the 300 mg/kg group. Osmotic Fragility of Erythrocytes:There were no biologically or statistically significant differences in osmotic fragility among the dose groups. The data were confounded by the presence of up to 54% reticulocytes and 12% Heinz bodies in the circulating blood in the 300 and 100 mg/kg groups. Division of the average hemolysis by the percentage of erythrocytes that were not reticulocytes and by the percentage of erythrocytes that did not contain Heinz bodies reduced the difference among the 300, 100 and 0 mg/kg groups. Electrophoresis of Hemoglobin: Electrophoresis of hemoglobin from the four dose groups produced seven peaks. These peaks were compared by analysis of migration distance from the origin (peak 0 overlays the origin, Table 10) and by the percentage of the total hemoglobin under each peak. There were no significant differences between sexes for any of the peaks or among the dose groups for peak 1. The percentage of total hemoglobin in peaks 2, 4 and 6 and their migration distance were compound and dose related. In general the no-effect dose for effects on the electrophoresis of hemoglobin was 30 mg/kg. However the no-effect dose for the increase in migration distance (mm) of peaks 4 and 5 was 100 mg/kg. The no-effect dose for the decrease in the percentage of total hemoglobin of peaks 0 and 3 was extrapolated t0 be 10 mg/kg.Correlation of Effects on Erythrocytes: Compound-related effects on erythrocytes included reduced concentration of hemoglobin and erythrocytes in circulating blood, reduced hematocrit, increased incidence of reticulocytes, Heinz bodies, polychromasia, macrocytosis anc siderocytes, increased migration of hemoglobin electrophoresis peaks 5 and 6, and increased percentage of total hemoglobin in peaks 2, 4 and 6. The combination of these effects was especially prevalent in 6 of the 20 surviving rats in the 300 mg/kg group and are characteristic of hemolytic anemia.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were no significant differences in the mean value of serum chemistry observations among the dose groups. The concentrations of aspartate aminotransferase and to a lesser extent of alanine aminotransferase, creatinine and urea nitrogen in sera from male rats of 100 mg/kg) and 300 mg/kg were considerably greater than the concentrations of these parameters in any other sera.Serum Electrophoresis: Observations from electrophoresis of serum included the concentration (g/dL) of total protein, albumin and alpha 1, alpha 2, beta and gamma globulins. The albumin/ globulin ratio (g/g) and the percentage of total protein of albumin and of each of the globulins were calculated. There were no significant compound or dose related effects among the dose groups.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Increased concentration of N-acetyl-glucosaminidase (NAG) and y-glutamyltransferase (GGT) in urine per mg creatinine were compound and dose related. The differences between the 0 and 30 mg/kg groups were not significant.Compound and dose related effects in urine included decreased urine volume and pH, increased specific gravity, increased concentration of protein, increased incidence of red and white blood cells per high power microscopic field of urine sediment, green to brown color and a hazy appearance. Effects in the 100 mg/kg group were minimal to minor and those in the 300 mg/kg group were moderate. There were no significant differences between the 0 mg/kg and 30 mg/kg groups. However, there were compound related trends in specific gravity, volume, concentration of protein and incidence of erythrocytes and leukocytes per high power field. There were no significant differences among the dose groups in any of the other components of the urinalysis.The concentration of calcium ions in the supernate from the 300 mg/kg group was ten times greater than in the 0 mg/kg group. The concentration of sodium, potassium, chloride, phosphate, sulfate and sulfite ions were similar in supernates from the 300 and 0 mg/kg groups.Effects of the test compound on urine were decreased volume, increased acidity and specific gravity, green brown discoloration; renal loss of protein, calcium, erythrocytes, leukocytes, NAG and GGT and excretion of the parent compound and its metabolites. The combination of these effects are characteristic of tubular nephrosis. They were especially prominent in four of the eight urine samples from the 300 mg/kg group and in one of the eight from the 100 mg/kg group.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute and/or relative weights of the liver, kidneys, spleen and heart were compound and dose-related. There were no other statistically or biologically significant differences among the dose groups.The absolute and relative weights of the spleens of the males and females in the 100 mg/kg and 300 mg/kg group were biologically and statistically much greater than those in the 0 and 30 mg/kg groups. The relative weights of the livers and kidneys of the males andfemales in the 300 mg/kg group and the absolute liver and kidney weights of the females in the 300 mg/kg group were statistically and biologically greater than the weights from the 0, 30 or 100 mg/kg group.The absolute and relative weights of the hearts from the females in the 300 mg/kg group were statistically greater than those in the 0, 30 or 100 mg/kg group. The increases in absolute and relative heart weight were not biologically significant and did not occur in the males.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross pathology included green and dark discoloration of the kidney and dark discoloration and enlargement of the spleen in the 300, 100 and 30 mg/kg group.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Hepatic extramedullary hematopoiesis and hypertrophy of hepatocytes; renal tubular pigmentation, hemoglobinuric nephrosis and regeneration; and splenic extramedullary hematopoiesis, hemosiderosis and congestion were observed. Hemoglobinuric nephrosis occurred in 36 of 40 rats in the 300 mg/kg group and in 33 of the 40 in the 100 mg/kg group. Renal tubular necrosis occurred in the 27 rats that died or became moribund during the exposure period. Nephrosis was minor to moderate in the surviving rats in the 300 mg/kg group, minimal to severe in the 100 mg/kg group and absent in the 30 and 0 mg/kg groups. The lesions consisted of amorphous brownish-red casts in the proximal convoluted tubules and degeneration of the tubular epithelial cells. Regenerative tubular epithelial cells in the distal convoluted tubules occurred in 37 of the 40 rats in the 300 mg/kg group, in 36 of the 40 in the 100 mg/kg group and in 18 of the 20 males in the 30 mg/kg group. These effects were minor to moderate in the100 mg/kg and 300 mg/kg groups, minimal to minor in the males in the 30 mg/kg group and absent in the females in the 30 mg/kg group and in both males and females in the 0 mg/kg group.Pigmentation of the proximal convoluted tubules with golden-brown granules occurred in 30 of the 40 rats in the 300 mg/kg group and in13 (12 females) of 40 in the 100 mg/kg group. These effects were minor to severe in the 300 mg/kg group, minimal to moderate in the 100 mg/kg group and did not occur in the 30 or 0 mg/kg groups. Extramedullary hematopoiesis occurred in the liver and/or spleen of 25 (16 male, 9 female) of 39 rats in the 300 mg/kg group and in 21 (13 male, 8 female) of 40 in the 100 mg/kg group. These lesions were minimal to minor in the liver and minimal to moderate (males only) in the spleen. They did not occur in the 30 mg/kg or 0 mg/kg groups.Hemosiderosis occurred in the liver and/or spleen in 35 of 40 rats in the 300 mg/kg group, in 35 of 39 in the 100 mg/kg group, in 17 of 20 males in the 30 mg/kg group and in 9 of 20 males in the 0 mg/kg group. Hemosiderosis was minor to severe in the 300 mg/kg and 100 mg/kg groups, and minimal to minor in the males ia the 30 mg/kg and 0 mg/kg groups. Hemosiderosis did not occur in the females in the 30 and 0 mg/kg groups. Hypertrophy of hepatocytes occurred in 13 of 20 female and 3 of 20 male rats in the 300 mg/kg group and in 4 of 20 female and 1 of 20 male rats in the 100 mg/kg group. These lesions were minor to moderate in the 300 mg/kg group, minor in the 100 mg/kg group and absent in the 30 mg/kg and 0 mg/kg groups.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

No biologically significant alterations were observed in blood pressure, heart rate or eletrocardiograms in any of the rats tested.

Effect levels

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table: Abnormal Clinical signs

Clinical signs	0	30	100	300	Total
	Male-female	Male-female	Male-female	Male-female
Compound related signs
Green brown discoloration of urine	None	36-45	52-50	52-54	289+
Abnormal activity: decreaed (71)* , lethargy (3), poor frooming (6)	1-0	None	0-1	37-41	80
Moribund (3) or dead (25)	0-1	None	2-5	9-11	28
Poor condition	None	Nobe	None	0-1	1
Subtotal- compound related signs	1-4	36-45	54-56	98-107	398
Sporadic signs**	5-1	7-2	4-4	4-6	33
Total	6-2	43-47	58-60	102-113	431

 

Table: Time and mortalty or morbundity in 27 rats in the 100 and 300 mg/Kg group (20/sex/group)

Exposure days	Doses Admin	100 mg/Kg		300 mg/Kg		Total
		Male	Female	Male	Female
1-15	1-12	0	2	0	1	3
16-30	13-23	1	1	2	9**	13
31-70	24-49	1	2	1	1**	5
71-94	50-66	0	0	6**	0	6
Total		2	5	9	11	27

                                             

Table: Mean average body weight by sex and dose group

Dose group (mg/Kg)	Mean average body weight
	Males	Females
300	389.7	218.4
100	416.5	231.8
30	416.7	237.9
0	409.0	234.6

 

Table: Hematologic parameters with statistically significant differences among dose groups

Dose group (mg/Kg)	Males				Females
	0	30	100	300	0	30	100	300
Hemoglobin (g/dL)	15.3	14.9	13.4	11.7	14.8	14.4	13.3	11.5
Hematocrit (%)	47.9	47.1	42.8	35.8	46.9	45.7	42.1	34.6
RBC (10^6/mm^3)	8.3	7.9	7.1	5.7	7.5	7.3	6.7	5.0
MCV (µ^3)	58.0	59.6	60.6	62.8	62.1	63.0	63.0	70.1
MCH (µµg)	18.5	18.8	19.0	20.6	19.6	19.8	19.8	23.4
MCHC (%)	31.8	31.5	31.4	32.7	31.5	31.5	31.6	33.2
Platelets (10^3/mm^3)	823	862	1034	1214	991	938	1037	1203
Reticulocytes (/1000 RBC)	9.9	13.3	33.4	113.4	133	15.4	31.2	221.3
Heinz bodies (/1000 RBC)	0.0	0.4	12.9	79.5	0.1	0.0	0.3	61.3

 

Table: Blood cell morphology

Dose group (mg/Kg)	Males				Females
	0	30	100	300	0	30	100	300
No. of observations (No./No. of survivors)	10/20	10/20	10/18	11/11	10/19	10/20	12/15	9/9
Normal (no. of rats)	7	1	0	0	7	2	0	0
Poikilocytosid No. affected severity	3 1,2	9 1,2	10 1,2	88 1,2,3	3 1,2	7 1,2	8 1	6 1,2,3
Polychromasia No. affected severity	0	0	9 1,2	11 3,4	0	2 1	11 1,2	9 3,4
Macrocytosis No. affected severity	0	0	8 1,2	11 2,3,4	0	1 1	10 1,2	9 3,4
Anisocytosis No. affected severity	0	0	8 1,2	11 2,3	0	1 1	10 1,2	9 1,2,3
Howell-jolly bodies No. affected severity	0	0	4 1	9 1	0	1 1	4 1	9 1
Siderocytes No. affected severity	0	0	3 1	7 1,2	0	0	5 1	9 1,2

 

Table: Percent hemolysis by osmotic gradient

Percent soln. (NaCl)	Males				Females
	0	30	100	300	0	30	100	300
.60	1.4	1.7	1.6	1.7	4.6	2.1	1.4	4.4
.55	6.6	8.2	6.4	5.9	18.3	11.9	6.7	11.4
.50	32.4	37.6	28.6	26.5	54.2	51.1	33.4	39.1
.45	69.5	77.5	68.0	65.2	83.6	84.0	74.3	78.4
.40	92.2	92.3	94.3	90.1	97.2	97.9	94.7	94.8
Average hemolysis (.55, .50 and .45% NaCl)	31.2	41.1	34.3	32.5	52.0	49.0	38.1	43.0
Percent normal erythrocytes (without reticulocytes or Heinz bodies)	99	99	95	82	99	98	87	73
Average hemolysis divided by percentage normal erythrocytes	31.5	41.5	36.1	39.6	52.5	50.0	43.8	58.9

 

Table: Urine chemistry tests

Dose Group (mg/Kg dose)	Males and females combined
	0	30	100	300
NAG (U/mg creatinine)
Mean	84	161	243	489
SD	25	98	85	256
GCT (U/mg creatinine)
Mean	23	35	78	109
SD	20	22	55	87

 

Table: Urinanalysis and observations of urine sediment

Dose Group (mg/Kg dose)	Males and females combined
	0	30	100	300
Specific gravity -mean	1.030	1.040	1.040	1.048
Volume, mL - mean	24.3	19.2	17.5	16.5
-largest	46.6	30.6	27.1	21.5
Protein 3+, 4+, >4+ (No. rats) 2+ <1+, 1+	0 3 5	0 6 2	0 7 1	4 4 0
pH 6.0,6.5 7.0, 7.5 8.0, 8.5	0 3 5	0 3 5	1 4 3	4 4 0
RBC/HPF <2 2-5 >5	5 2 0	5 1 2	6 1 1	3 3 2
WBC/HPF <20 20-50 >50	7 0 0	4 3 0	1 6 1	4 2 2
Color, green-brown (No. rats) yellow	0 8	0 8	3 5	8 0
Appearance Hazy (translucent) Cloudy (opaque)	0 8	0 8	1 1	3 5

 

Table: Analytical characteristics of supernates of urine from rats in the 0 and 300 mg/Kg group

Observation	3% aq. Solution of compound	Urine from 0 mg/Kg group	Urine from 300 mg/Kg group
UV absorbance
218 nm	Yes	No	No
270 nm	Yes	No	No
236 nm	No	No	Yes
HPLC
270 nm	Peaks at 5.6 and 11.1 min	No peaks	No peaks
236 nm	No peaks	No peaks	Peaks at 3.8 min
Mass spectrometry
Acidic extract	-	No	Phenylacetic acid
Basic extract	-	No	Methylaminophenol, aminophenol, brownish color

 

Table: Absolute and relative weights of organs with compound related effects, means of dose group by sex

Dose group (mg/Kg)	Males				Females
	0	30	100	300	0	30	100	300
Liver (g)	13.53	13.75	13.93	15.04	7.69	7.67	8.02	9.10*
% bw	2.83	2.85	2.86	3.27*	3.09	3.05	3.18	3.70*
Kidneys (g)	3.52	3.51	3.42	3.74	1.94	1.93	2.03	2.23*
% bw	0.74	0.73	0.70	0.82	0.78	0.77	0.81	0.91*
Spleen (g)	0.88	0.91	1.13*	1.87*	0.49	0.51	0.72*	1.30*
% bw	0.18	0.19	0.23*	0.40*	0.20	0.20	0.29*	0.53*
Heart (g)	1.54	1.57	1.60	1.60	0.91	0.92	0.91	1.00***
% bw	0.32	0.33	0.33	0.35	0.37	0.37	0.36	0.41***

* Statistically different from mean of 0 mg/kg gp.

**Not biologically significant

Applicant's summary and conclusion

Conclusions:

NOAEL for Elon is considered to be 30 mg/kg bw in male and female rats since toxic effects was observed at higher dosage concentrations.

Executive summary:

A repeated dose oral toxicity study was performed to determine the toxic nature of Elon upon repeated exposure for 91-94 days. Male and female Crl: COBS®, CD®, SD, BR rats were administered the test material by intubation five days a week with a dose of 0, 30, 100 or 300 mg/kg bw. The animals were observed for mortality, morbidity, clinical signs, body weight and organ weight changes, food consumption, hematology, serum chemistry, urinalysis, gross and histopathological parameters. Compound and dose-related effects occurred primarily in the 300 and 100 mg/kg groups and sporadically in the 30 mg/kg group. The major effects were degeneration of hemoglobin in circulating erythrocytes, hemolytic anemia, hemoglobinuric nephrosis and death. Compound-related morphologic effects in erythrocytes were slight (100 mg/kg group) to moderate (300 mg/kg group) and included polychromasia, macrocytosis, anisocytosis, siderocytosis and incidence of Howell-Jolly bodies. In addition, minimal to minor hypochromasia, microcytosis, spherocytosis and incidence of target cells occurred in 3 of 22 rats observed in the 100 mg/kg group and in 8 of 20 observed in the 300 mg/kg group. Compound and dose-related effects in urine included decreased urine volume and pH, increased specific gravity, increased concentration of protein, increased incidence of red and white blood cells per high power microscopic field of urine sediment, green to brown color and a hazy appearance. Effects in the 100 mg/kg group were minimal to minor and those in the 300 mg/kg group were moderate. Hepatic extramedullary hematopoiesis and hypertrophy of hepatocytes; renal tubular pigmentation, hemoglobinuric nephrosis and regeneration; and splenic extramedullary hematopoiesis, hemosiderosis and congestion were observed. Hemoglobinuric nephrosis occurred in 36 of 40 rats in the 300 mg/kg group and in 33 of the 40 in the 100 mg/kg group. Renal tubular necrosis occurred in the 27 rats that died or became moribund during the exposure period. Regenerative tubular epithelial cells in the distal convoluted tubules occurred in 37 of the 40 rats in the 300 mg/kg group, in 36 of the 40 in the 100 mg/kg group and in 18 of the 20 males in the 30 mg/kg group. These effects were minor to moderate in the 100 mg/kg and 300 mg/kg groups, minimal to minor in the males in the 30 mg/kg group and absent in the females in the 30 mg/kg group and in both males and females in the 0 mg/kg group. Extramedullary hematopoiesis occurred in the liver and/or spleen of 25 (16 male, 9 female) of 39 rats in the 300 mg/kg group and in 21 (13 male, 8 female) of 40 in the 100 mg/kg group. They did not occur in the 30 mg/kg or 0 mg/kg groups. Hemosiderosis occurred in the liver and/or spleen in 35 of 40 rats in the 300 mg/kg group, in 35 of 39 in the 100 mg/kg group, in 17 of 20 males in the 30 mg/kg group and in 9 of 20 males in the 0 mg/kg group. Hemosiderosis did not occur in the females in the 30 and 0 mg/kg groups. Hypertrophy of hepatocytes occurred in 13 of 20 female and 3 of 20 male rats in the 300 mg/kg group and in 4 of 20 female and 1 of 20 male rats in the 100 mg/kg group. Therefore, NOAEL of Elon was considered to be 30 mg/kg bw in male and female rats since higher dosage levels induced toxic responses.