Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
other: the study was performed with the sulfate salt of 1,4-diamino-2-methoxymethylbenzene
Adequacy of study:
key study
Study period:
From Mar. 13, 2006 to Jan. 07, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
according to Swiss Ordinance relating to GLP
Limit test:
no

Test material

Constituent 1
Reference substance name:
337906-37-3
Cas Number:
337906-37-3
IUPAC Name:
337906-37-3
Constituent 2
Reference substance name:
-
EC Number:
474-270-7
EC Name:
-
IUPAC Name:
474-270-7
Constituent 3
Reference substance name:
1,4-Diamino-2-methoxymethyl-benzene sulfate (1:1)
IUPAC Name:
1,4-Diamino-2-methoxymethyl-benzene sulfate (1:1)
Details on test material:
- Name of test material: 1,4-diamino-2-methoxymethyl-benzene sulfate (1: 1), (Methoxymethyl-PPD-Sulfat.) (code # A012220)
- TSIN: 801337 
- Molecular formula: C8C12N2OxH2SO4
- Molecular weight: 250.28
- Substance type: Pure active substance 
- Physical state: Cream-rosy powder
- Stability: The substance on storage in dryness and darkness is considered to be stable for more than 8 years. 
- Stability in Solution: Stable for seven days in water, DMSO, water and water/acetone 1:1 at room temperature
- Storage condition of test material: Away from direct sunlight at room temperature (15 -25 °C) 
- Solubility: The solubility in different solvents was as follows:
> 10 weight% in water (pH 1.75)
0.03 weight% in acetone
1.7 weight% in acetone/water (1:1)
15 weight% in DMSO

Test animals

Species:
rat
Strain:
other: HanRcc: WIST (SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS 
- Source: Harlan Laboratories Ltd., Laboratory Animal Services, 4414 Füllinsdorf / Switzerland
- Age at study initiation: 6 wk 
- Weight at study initiation: Male: 126.4 - 150.7 g (mean: 137.1 g) ; Female: 109.2 - 129.7 g (mean: 121.3 g) 
- Fasting period before study: No
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding 
- Diet: Pelleted standard Provimi Kliba 3433 (batch nos. 01/06, 76/05) rat maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland), ad libitum; The feed batches were analyzed for contaminants. 
- Water: Community tap-water from Itingen; ad libitum. (representative samples were analysed for bacteriological assay, chemical and contaminant) 
None of the contaminants analyzed in the water and diet is considered to have been present at a concentration that would have affected the validity of the results.
- Acclimation period: 7d

ENVIRONMENTAL CONDITIONS 
- Temperature: 22 ± 3 °C
- Relative humidity: 30-70% 
- Air changes: 10-15 air changes per h
- Photoperiod: 12 h light/12 h dark cycle per d (animals were exposed to fluorescent light with music during light period)

IN-LIFE DATES: From: Mar. 20, 2006 To: July 17, 2006

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.4% aqueous solution of ascorbic acid, adjusted with NaOH (1N) to pH 5.7 - 6.1
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared daily, test substance was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (15 – 25 °C). The dose formulations were adjusted with NaOH (1 N) to pH 5.7 – 6.1. Homogeneity of the test substance in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: Not Reported 
- Concentration in vehicle: 0, 1, 3 and 9 mg/mL
- Amount of vehicle: 10 mL/kg bw 
- Batch no.: Ascorbic acid: Fluka AG; batch # 1210661/5490565, 421110/1 12901 
Vehicle formulation: Ascorbic acid was weighed into a glass beaker (corrected for purity with a factor of 1.0375) on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (15 - 25°C).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity (collected from the top, middle and bottom of the mixing beakers) and stability (after 4 h) of the formulations were determined in samples taken after experimental start and at the end of the study. The analyses were performed by HPLC methods using a cation exchange column.

The results were as follows:
- The mean concentration for each dose formulation was within the accepted range of ±20% of the nominal content.
- Top, middle and bottom results for each concentration did not deviate by more than 8.7% (<15%) from the corresponding mean representing the homogeneity of the test substance.
- Stability (4 h) was demonstrated by the fact that results for each concentration did not deviate by more than 6.6% from the corresponding mean.
This confirms the correct preparation and storage of test substance formulations during the conduct of this study.
Duration of treatment / exposure:
90 d
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 30 and 90 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
- 15 males and 15 females (control and high dose group); 5 males and 5 females from each group were maintained as satellite (recovery) group 
- 10 males and 10 females (Low and mid dose group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based upon the results of a non-GLP 28-day dose range- finding study (RCC Study Number A17908) in which test substance was administered by gavage to 5 rats per group and sex.
- Rationale for animal assignment: Computer-generated random algorithm 
- Rationale for selecting satellite groups: Not reported
- Post-exposure recovery period: 27/28 d
Positive control:
no

Examinations

Observations and examinations performed and frequency:
MORTALITY: Yes
- Time schedule: Twice daily

CAGE SIDE OBSERVATIONS: Yes 
- Time schedule: Once daily during treatment and recovery
- Cage side observations included: Behavior, posture, kinked tail apex, skin / fur, fissures, ulcer and emaciated, secretion / excretion

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once weekly (Weeks 1-12 and 14-16) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during acclimatization and treatment periods, and immediately before necropsy. Body weights were recorded weekly during the recovery period.

FOOD CONSUMPTION: Yes
- The food consumption was recorded once during the acclimatization period and weekly thereafter, using an on-line electronic recording system

OPHTHALMOSCOPIC EXAMINATION: Yes 
- Time schedule for examinations: During acclimatization and at Week 13 and 17
- Dose groups that were examined: All 
- Procedure: The ophthalmoscopic examinations of both eyes of all animals was performed after the application of a mydriatic solution (Ciba Vision AG, 3172 Niederwangen / Switzerland) using a Miroflex 2 Ophthalmoscope (Eisenhut Vet. AG, 4123 Allschwil / Switzerland). A description of any abnormality was recorded. For unilateral findings unless otherwise indicated in the tables, the contralateral eye was without abnormalities.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 13 wks (Allocation A and B) and 17 wks (Allocation B)
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia) 
- Animals fasted: Yes (Animals were fasted in metabolism cages for approx. 18 h before blood sampling for laboratory investigations) 
- How many animals: All 
- Parameters examined: Erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hemoglobin concentration distribution width, platelet (thrombocyte) count, reticulocyte count, reticulocyte maturity index, methemoglobin, total leukocyte count, differential leukocyte count, thromboplastin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 13 wks (Allocation A and B) and after 17 wks (Allocation B)
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia) 
- Animals fasted: Yes (Animals were fasted in metabolism cages for approx. 18 h before blood sampling for laboratory investigations) 
- How many animals: All 
- Parameters examined: Glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, glutamate dehydrogenase, creatine kinase, alkaline phosphatase, gamma-glutamyl-transferase, sodium, potassium, chloride, calcium, phosphorus inorganic, total protein, albumin, globulin and albumin/globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: After 13 (Allocation A and B) and 17 wks (Allocation B). Urine was collected during the 18-h fasting period into a specimen vial. 
- Metabolism cages used for collection of urine: Yes
- Parameters examined: Volume (18 h), osmolality, specific gravity (relative density), color, appearance, pH, nitrite, protein, glucose, ketones, urobilinogen, bilirubin, erythrocytes and leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes 
- Time schedule for examinations: During Weeks 13 and 17 
- Dose groups that were examined: All during Week 13; satellite group of animals during Week 17 
- Battery of functions tested: Grip strength and locomotor activity

Sacrifice and pathology:
SACRIFICE: All animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and euthanised by exsanguination.

GROSS PATHOLOGY: Yes, all animals were weighed and necropsied after Week 13 (Allocation A) and Week 17 (Allocation B or satellite group). Descriptions of all macroscopic abnormalities were recorded.

- Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution or as indicated below:
Adrenal glands, aorta, bone (sternum, femur including joint), bone marrow (femur), brain (4 levels), cecum, colon, duodenum, epididymides (fixed in bouin's solution), esophagus, eyes with optic nerve (fixed in davidson's solution), harderian gland (fixed in davidson's solution), heart, ileum, with peyer's patches, jejunum with peyer's patches, kidneys, larynx, lacrimal gland (exorbital), liver, lungs (infused with formalin at necropsy), lymph nodes (mesenteric, mandibular), mammary gland area nasal cavity, ovaries, pancreas, pituitary gland, prostate gland (incl coagulating gland), rectum, salivary glands (mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, midthoracic, lumbar), spleen, stomach, testes (fixed in bouin's solution), thymus, thyroid (incl. parathyroid gland), tongue, trachea, urinary bladder (infused with formalin at necropsy), uterus, vagina and gross lesions

HISTOPATHOLOGY: Yes, all organ and tissue samples fixed in necropsy were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers, and stained with hematoxylin and eosin. Slides of all organs and tissues were examined by a pathologist.
Other examinations:
ABSOLUTE AND RELATIVE ORGAN WEIGHTS: Brain, thymus, spleen, heart, kidneys, testes, liver, adrenals, epididymides, thyroids, parathyroids, uterus and ovaries were weighed on schedule day of necropsy. The organ to terminal body weight (immediately prior to necropsy ) ratios as well as organ to brain weight ratios were determined
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity, ophthalmoscopic examinations, body weight, clinical laboratory investigations, organ weights and ratios, or macroscopic findings:
- The Dunnett-test (many to one t-test, or T-test, as appropriate) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution.
- Fisher's exact-test

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: At 30 mg/kg bw/day and at 90 mg/kg bw/day, orange discoloration of the urine was noted in males and females. Salivation and burrowing were reported at 30 mg/kg bw/day and 90 mg/kg bw/day.

BODY WEIGHT AND WEIGHT GAIN: Isolated instances of emaciated animals were observed. However, no differences in the mean body weights or mean body weight gain were noted during the treatment or recovery phases.

FOOD CONSUMPTION: No differences in the mean daily food consumption or mean relative food consumption were noted during the treatment phases and no late effects were noted during recovery.

OPHTHALMOSCOPIC EXAMINATION: Corneal opacities were reported in all groups including the controls at a frequency greater than historical control data for similarly aged Wistar rats. These findings were considered to be related to localized irritation by foreign material (e.g., bedding) as a consequence of burrowing. As there were no correlating histopathological findings, this in-life observation was considered to be of no toxicological relevance.

HAEMATOLOGY, CLINICAL CHEMISTRY AND URINALYSIS: In the high dose group animals, minor changes were observed in haematological and biochemistry values. All of these values were within the range of historical reference control values. The observed changes in the urine values in the high dose animals were minor and none attained statistical significance. In addition, both for the haematological and biochemistry as well as for the urine values no accompanying histopathological changes were observed in the target organs. The reported findings in organ weights (elevated relative kidney weight in high dose females and elevated heart weight in low dose females) were incidental and not accompanied by any histopathological changes. Some incidental histopathological findings (renal tubular basophilia, renal mineralization, luminal dilation of the rectum) were not dose-related and were similar in incidence to the historical control data. Therefore, all of the above mentioned findings were considered of no toxicological relevance.
In high dose: An increase in absolute and relative liver weights was observed only in males and was minor in magnitude (8% for relative liver weight). Aspartate aminotranserfase (AST) was elevated in males (statistically not significant). Lactate dehydrogenase (LDH) levels were increased in males and females (achieved statistical significance), but were well within the range of the historical control database. The creatine kinase (CK) elevation observed in both sexes (and with statistical significance in males) was well within the range of the historical controls. AST and LDH occur in many body tissues and are not organ-specific enzymes. CK is not an indicator of hepatoxicity since CK isozymes are found in skeletal and cardiac muscle. Therefore, in the absence of specific hepatic enzyme markers (e.g. alanine aminotransferase (ALT), alkaline phosphatase (ALP)), the marginal elevations in AST, LDH and CK cannot be considered as an indicator of hepatotoxicity. The minor observed increase in liver weight was not associated with pathological findings or liver specific serum enzyme alterations and was therefore considered to be adaptive in nature. Minimal hepatocellular hypertrophy was noted in one male animal. In the absence of significant hepatic changes (necrosis, inflammation) or increases in specific hepatic enzyme markers in the serum, the minimal hypertrophy in one male was concluded to be adaptive. Overall, the enzyme changes in AST, LDH, and CK and the liver weight elevation in the males were minor, mostly within the normal historical range, and were not indicative of organ toxicity, especially in the absence of significant histopathological changes (neither found in the liver, nor in other organs).

NEUROBEHAVIOUR: The mean hind limb grip strength value of high dose group males was significantly lower than those of the controls after the treatment period and also after 4 wks of recovery. In mid dose group males fore limb grip strength value was significantly elevated and the hind limb grip strength was significantly reduced after the end of the treatment period. Since the findings were not dose dependent, were not consistent between sexes in the same dose group, and were of different character (decrease and increase), these deviations in the grip strength, which are commonly observed in rats, were considered as not test material related.
- Variations in the locomotor activity were observed in females of low and mid dose group and males and females of high dose group. However, these changes showed neither a dose dependency, nor a consistency of the character (increase vs. decrease). Therefore, these findings were not considered to be treatment related.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
90 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Overall effects
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Based on minimal liver-related findings in high dose group.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Methoxymethyl-PPD-Sulfate was administered by oral gavage at dose levels of 0, 10, 30 and 90 mg/kg bw/day for 13 wk. The minimal liver-related findings observed in males at the high dose level (90 mg/kg bw/day) were considered to be adaptive responses and not to be of toxicological relevance. Therefore, 90 mg/kg bw/day was concluded to be the no observed adverse effect level (NOAEL) for 2- Methoxy-Methyl-P-Phenylenediamine Sulfate.
In the absence of any changes compared to the control animal, the NOEL was concluded to be 30 mg/kg bw/day.
Executive summary:

The repeated dose toxicity study of 1,4-diamino-2-methoxymethyl-benzene sulfate (1: 1), (Methoxymethyl-PPD-Sulfat.) was determined following OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents). 

In this subchronic toxicity study,2-Methoxy-Methyl-P-Phenylenediamine Sulfate was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 10, 30 and 90 mg/kg body weight/day for a period of 90 days. A control group was treated similarly with the vehicle (0.4% aqueous solution of ascorbic acid).

The groups comprised 10 animals per sex, which were sacrificed after 90 days of treatment. Additional 5 rats per sex and group were used at 0 and 90 mg/kg. These animals were treated for 90 days and then allowed a 28-day treatment-free recovery period after which they were sacrificed.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pre-test, the treatment and recovery periods. Ophthalmoscopic examinations were performed at pre-test, the end of the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during Week 13. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for haematology and plasma chemistry analyses and urine samples were collected for urinalyses.

All animals were euthanized, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and treated animals in both the main study and the recovery groups. Analytical results indicate that dosing solutions met the acceptance criteria for concentration of test substance

All animals survived until scheduled necropsy. At 30 mg/kg bw/day and at 90 mg/kg bw/day, orange discoloration of the urine was noted in males and females. Salivation and burrowing were reported at 30 mg/kg bw/day and 90 mg/kg bw/day.

Corneal opacities were reported in all groups including the controls at a frequency greater than historical control data for similarly aged Wistar rats. These findings were considered to be related to localized irritation by foreign material (e.g., bedding) as a consequence of burrowing. As there were no correlating histopathological findings, this in-life observation was considered to be of no toxicological relevance.

In the high dose group animals, minor changes were observed in haematological and biochemistry values. All of these values were within the range of historical reference control values. The observed changes in the urine values in the high dose animals were minor and none attained statistical significance. In addition, both for the haematological and biochemistry as well as for the urine values no accompanying histopathological changes were observed in the target organs. The reported findings in organ weights (elevated relative kidney weight in high dose females and elevated heart weight in low dose females) were incidental and not accompanied by any histopathological changes. Some incidental histopathological findings (renal tubular basophilia, renal mineralization, luminal dilation of the rectum) were not dose-related and were similar in incidence to the historical control data. Therefore, all of the above mentioned findings were considered of no toxicological relevance.

An increase in absolute and relative liver weights was observed only in males and was minor in magnitude (8% for relative liver weight). Aspartate aminotranserfase (AST) was elevated in males (statistically not significant). Lactate dehydrogenase (LDH) levels were increased in males and females (achieved statistical significance), but were well within the range of the historical control database. The creatine kinase (CK) elevation observed in both sexes (and with statistical significance in males) was well within the range of the historical controls. AST and LDH occur in many body tissues and are not organ-specific enzymes. CK is not an indicator of hepatoxicity since CK isozymes are found in skeletal and cardiac muscle. Therefore, in the absence of specific hepatic enzyme markers (e.g. alanine aminotransferase (ALT), alkaline phosphatase (ALP)), the marginal elevations in AST, LDH and CK cannot be considered as an indicator of hepatotoxicity. The minor observed increase in liver weight was not associated with pathological findings or liver specific serum enzyme alterations and was therefore considered to be adaptive in nature. Minimal hepatocellular hypertrophy was noted in one male animal. In the absence of significant hepatic changes (necrosis, inflammation) or increases in specific hepatic enzyme markers in the serum, the minimal hypertrophy in one male was concluded to be adaptive. Overall, the enzyme changes in AST, LDH, and CK and the liver weight elevation in the males were minor, mostly within the normal historical range, and were not indicative of organ toxicity, especially in the absence of significant histopathological changes (neither found in the liver, nor in other organs).

In conclusion, the minimal liver-related findings observed in males at the high dose level (90 mg/kg bw/day) were considered to be adaptive responses and not to be of toxicological relevance. Therefore, 90 mg/kg bw/day was concluded to be the no observed adverse effect level (NOAEL) for 2- Methoxy-Methyl-P-Phenylenediamine Sulfate.

In the absence of any changes compared to the control animal, the NOEL was concluded to be 30 mg/kg bw/day.

 

This subchronic toxicity study is classified as acceptable, and satisfies the guideline requirements of the OECD 408 method.