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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
red powder
Batch # : M266/4279

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 %
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test substance concentrations were performed colorimetrically. Deviations of +- 10 % of the target concentration were tolerated. Homogeneity and concentrations of the tested solutions of Hydroxyethyl-2-nitro-p-toluidine were analytically confirmed. The determined concentrations revealed mean values of 94 – 103 % of nominal. Thus, the nominal concentrations were used in the report. Investigation on homogeneity showed deviations of less than 5 %. Based on these results, a sufficient homogeneity in the chosen vehicle was demonstrated.
Duration of treatment / exposure:
4 consecutive weeks
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control group
Dose / conc.:
80 mg/kg bw/day (nominal)
Dose / conc.:
240 mg/kg bw/day (nominal)
Dose / conc.:
720 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control:
no

Examinations

Observations and examinations performed and frequency:
Mortality, clinical signs and behavioural changes were recorded once daily and individual body weights and food consumption were recorded weekly. A detailed ophthalmologic investigation was performed before treatment and at day 28. Clinical laboratory investigations (haematology, blood/clinical biochemistry) were performed with 5 animals per sex and dose group at day 0 and day 28 and additionally on day 42 for selected parameters. Urine was collected in metabolism cages over night between the days –1/0, 27/28 and additionally between days 41/42 (satellite groups).
Sacrifice and pathology:
All animals were subjected to a detailed necropsy. Adrenals, kidneys, liver and testes were weighed and various tissues and organs were fixed and stored for further examination if required. The following organs/tissues of the control and high dose animals were examined histopathologically: adrenal glands, kidney, liver, heart and spleen. In addition, all gross lesions noted in any organ were also examined microscopically.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The observed staining of fur, tail and bedding material noted for all dose groups is attributed to the dying properties of the test substance and is therefore not regarded as a toxic change.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight development was not affected by the treatment with Hydroxyethyl-2-nitro-p-toluidine. The food consumption, although significantly decreased if compared to the controls in high dose females, did not affect the body weight development. For the high dose males a significant increase in food consumption was noted during the recovery period only. However, this had no influence on the body weight development.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The ophthalmologic examination revealed no abnormalities
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group some haematological parameters were affected. In particular, mean corpuscular haemoglobin concentration (MCHC) and lymphocytes were decreased in males, whereas the number of neutrophils was elevated. In females the Hb (hemoglobin concentration) and the RBC- (erythrocyte count) values showed a decrease if compared to the control. No clear dose-dependent effect was noted with regard to the haematocrit value. A statistically significant decrease was seen in the mid dose females, but not in the high dose females, where only a slight decrease was observed. In the animals observed until the end of the recovery period, only the MCHC values in males were significantly different to the control.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant effects on clinical parameters were seen in the male high dose group only. Elevated levels for calcium, alkaline phosphatase, GOT, GPT and total bilirubin as well as decreased levels for glucose and cholesterol were noted. In females only, alkaline phosphatase was elevated, but without showing a statistically significant difference to the concurrent control. After the 2 week recovery period, the only noted statistically significant differences observed were elevated GOT and GPT levels in males.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urinalysis revealed a lower pH value for the high dose males (statistically significant) and for females (not statistically significant). The determination of other urinary parameters was compromised in some animals of all dose groups due to the intensive staining (dark yellow to dark red) of the urine. This discoloration of the urine was due to the renal excretion of the tested dye and was thus not considered as a toxic effect. However, it clearly demonstrates the bioavailability of Hydroxyethyl-2-nitro-p-toluidine after oral exposure. All other urine parameter besides colour and pH-value revealed no differences to the concurrent controls. The intensive colouring of the urine as well as the lower pH value was still noted in the animals of high dose group at the end of the recovery period.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The weights of some organs varied in a statistically significant manner. However, the effect noted for kidney weights reveal no dose-response and no consistent alteration for the contra-lateral organ. Therefore those variations are considered as incidental. The effects noted in females of the mid dose group (lower relative adrenal weight) is most likely due to the high body weight of this group at this observation time point as all relative organ weights were lower in this group, but without reaching statistical significance. This variation is therefore also not considered as treatment related. In summary, the increase in the relative liver weights of high dose males is judged as substance related effect, as effects on marker enzymes for a potential liver damage were also noted in this group.
Description (incidence and severity):
The gross necropsy revealed some tissue staining especially for the high dose groups. In the mid and high dose males one animal revealed a red thymus; no other lesions were noted. For the recovery groups besides some external staining (fur and or tail) no lesions were observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The histopathological examination revealed no treatment related findings.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
The histopathological examination revealed no treatment related findings.

Effect levels

Dose descriptor:
NOAEL
Effect level:
240 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Treatment-related and toxicologically relevant effects (statistically significantly elevated liver weights and increased clinical chemistry values like GOT, GPT or alkaline phosphatase in males; decreased Hb- and RBC-values in females) were observed at the highest tested dose of 720 mg/kg bw/day. This was not the case for the next lower dose of 240 mg/kg bw/day. Therefore, a No Observed Adverse Effect Level (NOAEL) of 240 mg/kg bw/day was deduced from this 28-day oral toxicity study with Hydroxyethyl-2-nitro-p-toluidine.
Executive summary:

Doses of 80, 240, 720 mg/kg bw/day Hydroxyethyl-2-nitro-p-toluidine (dissolved in bi-distilled water containing 0.5 % CMC) were administered daily to groups of 5 male and 5 female Wistar Crl: (Wi)/Br (SPF) rats by gavage in a total volume of 10 ml/kg bw over a period of 4 consecutive weeks. A control group of equal size received the same dose volume of the vehicle (10 ml/kg, 0.5 % CMC) daily throughout the dosing period. For recovery observations, satellite groups of additional 5 males and 5 females for the control and the high dose groups, respectively, were investigated after a 2-week treatment-free period. Mortality, clinical signs and behavioural changes were recorded once daily and individual body weights and food consumption were recorded weekly. A detailed ophthalmologic investigation was performed before treatment and at day 28. Clinical laboratory investigations (haematology, blood/clinical biochemistry) were performed with 5 animals per sex and dose group at day 0 and day 28 and additionally on day 42 for selected parameters. Urine was collected in metabolism cages over night between the days –1/0, 27/28 and additionally between days 41/42 (satellite groups). All animals were subjected to a detailed necropsy. Adrenals, kidneys, liver and testes were weighed and various tissues and organs were fixed and stored for further examination if required. The following organs/tissues of the control and high dose animals were examined histopatho-logically: adrenal glands, kidney, liver, heart and spleen. In addition, all gross lesions noted in any organ were also examined microscopically. Homogeneity and concentrations of the tested solutions of Hydroxyethyl-2-nitro-p-toluidine were analytically confirmed. The determined concentrations revealed mean values of 94 – 103 % of nominal. Thus, the nominal concentrations were used in the report. Investigation on homogeneity showed deviations of less than 5 %. Based on these results, a sufficient homogeneity in the chosen vehicle was demonstrated.

No deaths and no treatment-related clinical signs were noted in any animal (besides salivation in the high dose group). The observed staining of fur, tail and bedding material noted for all dose groups is attributed to the dying properties of the test substance and is therefore not regarded as a toxic change. The body weight development was not affected by the treatment with Hydroxyethyl-2-nitro-p-toluidine. The food consumption, although significantly decreased if compared to the controls in high dose females, did not affect the body weight development. For the high dose males a significant increase in food consumption was noted during the recovery period only. However, this had no influence on the body weight development. The ophthalmologic examination revealed no abnormalities. In the high dose group some haematological parameters were affected. In particular, mean corpuscular haemoglobin concentration (MCHC) and lymphocytes were decreased in males, whereas the number of neutrophils was elevated. In females the Hb (hemoglobin concentration) and the RBC- (erythrocyte count) values showed a decrease if compared to the control. No clear dose-dependent effect was noted with regard to the haematocrit value. A statistically significant decrease was seen in the mid dose females, but not in the high dose females, where only a slight decrease was observed. In the animals observed until the end of the recovery period, only the MCHC values in males were significantly different to the control. Statistically significant effects on clinical parameters were seen in the male high dose group only. Elevated levels for calcium, alkaline phosphatase, GOT, GPT and total bilirubin as well as decreased levels for glucose and cholesterol were noted. In females only, alkaline phosphatase was elevated, but without showing a statistically significant difference to the concurrent control. After the 2 week recovery period, the only noted statistically significant differences observed were elevated GOT and GPT levels in males. Urinalysis revealed a lower pH value for the high dose males (statistically significant) and for females (not statistically significant). The determination of other urinary parameters was compromised in some animals of all dose groups due to the intensive staining (dark yellow to dark red) of the urine. This discoloration of the urine was due to the renal excretion of the tested dye and was thus not considered as a toxic effect. However, it clearly demonstrates the bioavailability of Hydroxyethyl-2-nitro-p-toluidine after oral exposure. All other urine parameter besides colour and pH-value revealed no differences to the concurrent controls. The intensive colouring of the urine as well as the lower pH value was still noted in the animals of high dose group at the end of the recovery period. The gross necropsy revealed some tissue staining especially for the high dose groups. In the mid and high dose males one animal revealed a red thymus; no other lesions were noted. For the recovery groups besides some external staining (fur and or tail) no lesions were observed.

The weights of some organs varied in a statistically significant manner. However, the effect noted for kidney weights reveal no dose-response and no consistent alteration for the contra-lateral organ. Therefore those variations are considered as incidental. The effects noted in females of the mid dose group (lower relative adrenal weight) is most likely due to the high body weight of this group at this observation time point as all relative organ weights were lower in this group, but without reaching statistical significance. This variation is therefore also not considered as treatment related. In summary, the increase in the relative liver weights of high dose males is judged as substance related effect, as effects on marker enzymes for a potential liver damage were also noted in this group. The histopathological examination revealed no treatment related findings.