Registration Dossier

Administrative data

Endpoint:
specific investigations: other studies
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From June 01, 1981 to June 30, 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented document, followed basic scientific principles and GLP
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report Date:
1981

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The effects of the test substance on thyroid function in 5 rats /sex were evaluated after three weeks (6 days/week) of oral exposure through daily clinical observations, weekly body weights measurements, terminal hematological and thyroid function evaluations. Gross pathology was also conducted on all organs, while histopathology was conducted only on the thyroid.
GLP compliance:
yes
Type of method:
in vivo
Endpoint addressed:
repeated dose toxicity: oral

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material: 1-Methyl-2, 5-diaminobenzene sulphate (Toluene-2,5-diamine sulfate)- Substance type: Pure active substance- Physical state: Fine, violet powder- Stability under test conditions: Stable for years, in closed containers and protected from light- Storage condition of test material: Not reported- pH: 6.5

Test animals

Species:
rat
Strain:
other: SPF Wistar TNO/W. 74
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Winkelmann- Age at study initiation: Not reported- Weight at study initiation: Males (198 - 221 g) and Females (144 - 171 g)- Housing: Animals were housed in groups of 5 male or 5 female rats in Macrolon cages, type III (Ebeco) on standard litter "bedding" (Ssniff). - Diet: Standard laboratory diet Number 1324 (Altromin). The composition of the standard diet is provided in the study report.- Water: Tap water, ad libitum- Acclimation period: 7 dENVIRONMENTAL CONDITIONS- Temperature : 20 ± 2°C- Humidity: 50± 5%- Air changes : 15x per hour- Photoperiod : 12 h dark /12 h lightEXPERIMENT INITIATION DATE: June 30, 1981EXPERIMENT COMPLETION DATE: June 30, 1981

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing solutions were prepared fresh each day and used immediately.VEHICLE (Water)- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
3 Wks
Frequency of treatment:
6 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:10 and 50 mg/kg Basis:analytical conc.
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose selection: The dose levels for the test substance were selected on the basis of its LD50 value for rats, such that both a dose-response relationship and a no-effect level could be attained.

Examinations

Examinations:
1. Clinical observations: Daily observations were made for mortality, motor behavior, production of urine and feces, and the appearance of the fur and body orifices. Clinical observations were registered on a weekly basis. 2. Body weights: Individual body weights were registered on a weekly basis with an electronic balance with digital display (Model MP 1364, Data Print, Sartorius). 3. Hematology: Blood samples were collected from all test animals and controls prior to culling at the end of the experiment. Erythrocytes, total leukocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean hemoglobin content (MCH) of single erythrocytes, mean corpuscular hemoglobin concentration (MCHC), and differential blood count were measured by staining according to Pappenheim and counting under microscope (Visopan, Reichert). 4. Thyroid hormone measurements: Total thyroxin levels (T4), Triiodothyronine (T3) and Thyroxin binding index (TBI) were measured. Free thyroxin index (FT4I) was calculated as T4/TBI. Enzyme linked immunosorbent assay (ELISA) was used for hormone measurements. 5. Gross pathology: The animals were culled at the end of the experiment by means of ether narcosis and subsequent exsanguinations through the axillary artery. All organs were macroscopically evaluated.6. Histopathology: Only thyroid slides were prepared and examined microscopically.

Results and discussion

Details on results:
CLINICAL OBSERVATIONS: Over the whole course of the experiment, all animals (treated groups and control) exhibited normal behavior, normal fur, and normal feces production. Examinations of eyes, hearing, and reflexes showed no deviations from the physiological norm. The clinical behavior of both treated and untreated groups did not show any anomalies. MORTALITIES: All animals survived the duration of the study. GROSS PATHOLOGY FINDINGS: There were no findings in the control group or the 10 mg/kg female group. Slight uniform dark discoloration of the thyroids was observed in 1/5 males in 10 mg/kg group and in 4/5 females in the 50 mg/kg group. Slight dark discoloration was observed in 5/5 males in the 50 mg/kg group.BODY WEIGHTS: Slightly increased body weight growth was observed in the 10 mg/kg females, when compared to controls. A highly significant reduction in body weight was observed in 50 mg/kg males. RED BLOOD CELL COUNTS: The hemoglobin content of individual erythrocytes at constant erythrocyte volume was highly significantly reduced in the 10 mg/kg dose group (males and females) when compared to control values. This finding was accompanied by a significant reduction of hemoglobin concentration of erythrocytes of respective groups; hypochromic anemia was diagnosed in these animals. WHITE BLOOD CELL COUNT: Neutrophils were slightly significantly increased in the 50 mg/kg females and highly significantly increased in the 10 mg/kg females. HISTOPATHOLOGY (Thyroid only): In the 10 mg/kg dose group, all animals except for one female showed normal thyroid structures. The epithelia of the affected female exhibited a flat-cubic shape and the colloid had a rose-red to gray-rose color. In the 50 mg/kg dose group, all animals except one male and one female showed normal thyroid structures. The affected animals exhibited flat-cubic epithelia, but all other characteristics were normal. These results were qualified by the relatively small number of animals per sex per dose group. THYROID HORMONE ANALYSES: The thyroxine binding index was weakly significantly reduced in males of the 50 mg/kg dose group. T3 levels were significantly reduced in males of the 50 mg/kg dose group.

Any other information on results incl. tables

In the control animals, the thyroids exhibited a normal structure both for male and female animals: the flat epithelia was of red-bluish color, the follicles were of middle size, and the abundant colloid was uniformly colored in red. Interfollicularly, there was a pronounced interfollicular solidification.

Applicant's summary and conclusion

Conclusions:
1-Methyl-2, 5-diaminobenzene sulphate (Toluene-2, 5 -diamine sulfate) when administered orally to SPF Wistar TNO/W. 74 rats at dose levels of 10 and 50 mg/kg bw for 3 weeks revealed a LOAEL at 10 mg/kg bw.
Executive summary:

The purpose of this study was to evaluate the effect of 1-Methyl-2, 5-diaminobenzene sulphate (Toluene-2,5 -diamine sulfate) on thyroid function in Wistar rats after three weeks (6 days/week) of exposure to the test substance via oral gavage.

SPF Wistar TNO/W. 74 rats weighing 198 - 221 g for male rats and 144 - 171 g for female rats
were used. The feed used was standard laboratory diet (ad libitum).The animals were housed in groups of 5 by sex in Macrolon cages, Type III (Ebeco). Standard laboratory conditions were maintained (temperature: 20±2°C; humidity: 50±5%; artificial light: 12 h cycle).

In this study, two dose levels of 10 and 50 mg/kg were tested using five males and five females per dose group. The control group of five males and five females received the vehicle, water.

The following parameters and end points were evaluated in this study: clinical signs, mortality, body weights, body weight changes, hematology, clinical chemistry, gross necropsy, histopathologic examinations of the thyroids with special emphasis on height and color of the gland's epithelia, size and structure of the follicles, and stainability of the colloid, and thyroid hormone analyses. Thyroid hormone measurements included the following: total thyroxin levels (T4), triiodothyronine (T3) and thyroxin binding index (TBI). Free thyroxin index (FT4I) was calculated as T4/TBI. Enzyme linked immunosorbent assay (ELISA) was used for hormone measurements.

All animals survived the duration of the study. There were no significant clinical observations. Slightly increased body weight growth was observed in the 10 mg/kg females, when compared to controls. A highly significant reduction in body weight was observed in 50 mg/kg males. The hemoglobin content of individual erythrocytes at constant erythrocyte volume was highly significantly reduced in the 10 mg/kg dose group (males and females) when compared to control values. This finding was accompanied by a significant reduction of hemoglobin concentration of erythrocytes of the respective groups. Hypochromic anemia was diagnosed for these animals. Neutrophils were slightly significantly increased in the 50 mg/kg females and highly significantly increased in the 10 mg/kg females. The thyroid hormone analyses showed that the thyroxine binding index and T3 levels were reduced in males of the 50 mg/kg dose group.

Gross pathology observations showed that there were no findings in the control group and the 10 mg/kg female group. Slight uniform dark discoloration of the thyroids was observed in 1/5 males in 10 mg/kg group and in 4/5 females in the 50 mg/kg group. Slight dark discoloration was observed in 5/5 males in the 50 mg/kg group. Histopathology observations in the 10 mg/kg dose group showed that all animals except for one female showed normal thyroid structures. The epithelia of the affected female exhibited a flat-cubic shape and the colloid had a rose-red to gray-rose color. In the 50 mg/kg dose group, all animals except one male and one female showed normal thyroid structures. The affected animals exhibited flat-cubic epithelia, but all other characteristics were normal. These results were qualified by the relatively small number of animals per sex per dose group.

Comparing the morphological, histomorphological, and clinical chemistry findings, it was observed that the morphological responses of the thyroid were different at two different doses of the test substance. In this comparison, an increased susceptibility of males compared to females was noticeable. The findings of stronger thyroid activation and numerical increasing trend in activation in male animals was consistent with the known higher susceptibility of male animals for such thyroid changes. The histomorphological changes were consistent with the gross macroscopy findings.

Based on above, it was concluded that 1-Methyl-2, 5-diaminobenzene sulphate (Toluene-2, 5 -diamine sulfate) when administered orally to SPF Wistar TNO / W. 74 rats at dose levels of 10 and 50 mg/kg bw for 3 weeks revealed a LOAEL (Low Observed Adverse Effect Level) at 10 mg/kg bw.