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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:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: although secondary citation from peer reviewed publication the given information is valuble and reliable to be considered

Data source

Reference
Reference Type:
publication
Title:
Urinary bladder - 14-day feeding study with o-toluidine in rats
Author:
DuPont 1994 cited by OECD
Year:
2006
Bibliographic source:
OECD/SIDS: o-toluidine (CAS-No.:95-53-4), DuPont: OTS0557449, published by UNEP, 2006

Materials and methods

Principles of method if other than guideline:
A sub-acute (14 days) feeding study in male and female Fischer rats (n = 5 per sex and group) with doses of 0, 500, 3000, 6000 ppm (corresponding to an estimated substance intake of 40.4, 238, 449or 43.5, 251, 481 mg/kg bw/day of males or females, respectively, was performed to investigate the urinary bladder toxicity by histopathological evaluation as well as by quantitative determination of the urinary metabolite N-acetyl-4-amino-m-cresol (NAAC) and the level of methemoglobinemia as potential biological exposure indices (DuPont, 1994).
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
o-toluidine
EC Number:
202-429-0
EC Name:
o-toluidine
Cas Number:
95-53-4
Molecular formula:
C7H9N
IUPAC Name:
o-toluidine
Test material form:
not specified
Details on test material:
content: 99.5 %

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: approx 59 d
- Weight at study initiation: males 179-196g; females 118-137 g
- Housing: 1 rat per cage
- Water ad libitum
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
daily 0, 500, 3000, 6000 ppm (corresponding to an estimated substance intake of 40.4, 238, 449or 43.5, 251, 481 mg/kg bw/day of males or females, respectively, not adjusted to
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
as reported by OECD/SIDS:
the test substance stability for 7 days: 58.6 % (500 ppm), 68 % (3000 ppm) and 66.8 % (6000 ppm)
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 500, 3000, 6000 ppm (corresponding to an estimated substance intake of 40.4, 238, 449or 43.5, 251, 481 mg/kg bw/day of males or females, respectively,
Basis:

No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
Details on study design:
Groups of 5 animals/sex/dose were fed different amounts of the test substance mixed in the diet for 14 days ; no post application observation period
Positive control:
no

Examinations

Observations and examinations performed and frequency:
as reported by OECD/SIDS:
Clinical signs and frequency:
clinical signs : during weighing (3 times per week)
Mortality twice daily during the week one daily on weekends
body weigfht yes 3 times per week
food consumption yes individually per week
Sacrifice and pathology:
as reported by OECD/SIDS:
Necropsy at the end of the treatment periodfor each amimal the bladder and sections of the duodenum were removed adn processed for the various toxicity evaluation (as appropriate and as positive control for the immunohistopathological stainingtechnique)
Histoapathological evaluation was limited to the urinary bladder
Other examinations:
as reported by OECD/SIDS:
blood sampling for methemoglobinanalyses immediate after sacrifice
urinary metabolite quantitation: N-acetyl-4-amino-m-cresol (NAAC)
Statistics:
as reported by OECD/SIDS:
bartlett's test, one way analysis of varilance, Dunnett's test, Least Significant difference, Cochran Armitrage test

Results and discussion

Results of examinations

Details on results:
---No mortality and no compound-related clinical findings were reported, except for wet and stained perineum in female rats at 6000 ppm after one week.

---Slight but statistically significantly decreased body weights were observed in the high dose groups (males: 218 g versus 233 g of controls; females: 142 g versus151 g of controls). Body weight gain was decreased in high dosed males (30.0 g versus 43.5 g of controls) and in all dose groups of females (low, mid and high dose: 19.2 g, 18.6 g and 15.4 g versus 23.5 g of controls).

--- Urothelial hyperplasia was described to be mild in the high dosed females, and in males, thickening of the urothelial layer was minimal (detailed data were not given) .Urinary bladder epithelial cell proliferation, characterized by labeling indices, increased with increasing dosage of o-toluidine but was significant only in the 6000 ppm dosed males (1.42 versus 0.47 of controls) and in the 6000 and 3000 ppm dosed females (2.55 and 0.38 versus 0.08 of controls).

---Urinary excretion of NAAC was positively correlated with the cell proliferation in both male and female rats, according to the author, indicating that NAAC may be a useful urinary biomonitor for o-toluidine exposure.

----Statistically significant and dose-related increase in methemoglobin production was determined in all treated animals (males: 4.2 %, 10.7 %, and 14.9 % versus 0.6 % in controls; females: 6.2 %, 14.5 %, and 19.0 % versus 0.5 % in controls).

Therefore a NOAEL could not be established. The LOAEL based on methemoglobinemia (male and females) and decreased body weight gain (females) was 500 ppm (adjusted to the test substance stability: app. 23.7 mg/kg bw/day for males and app. 25.5 mg/kg bw/day for females) (DuPont, 1994 cited in OECD/SIDS 2006).

Effect levels

Dose descriptor:
LOAEL
Effect level:
500 ppm
Sex:
male/female
Basis for effect level:
other: approx 37.5 mg/kg bw/day (corrected: 23.5 mg/kg bw/day): based on methemoglobinemia and decreased body weight gain in males and females

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

A sub-acute (14 days) feeding study in male and female Fischer rats (n = 5 per sex and group) withdoses of 0, 500, 3000, 6000 ppm (corresponding to an estimated substance intake of 40.4, 238, 449or 43.5, 251, 481 mg/kg bw/day of males or females, respectively, not adjusted to the test substancestability: 58.6 % (500 ppm), 68 % (3000 ppm) and 66.8 % (6000 ppm)was performed toinvestigate the urinary bladder toxicity by histopathological evaluation as well as by quantitativedetermination of the urinary metabolite N-acetyl-4-amino-m-cresol (NAAC) and the level ofmethemoglobinemia as potential biological exposure indices (DuPont, 1994). Additional 5 - 10 ratsper sex and group were used to perform an UDS test. No mortality and nocompound-related clinical findings were reported, except for wet and stained perineum in femalerats at 6000 ppm after one week. Slight but statistically significantly decreased body weights wereobserved in the high dose groups (males: 218 g versus 233 g of controls; females: 142 g versus151 g of controls). Body weight gain was decreased in high dosed males (30.0 g versus 43.5 g ofcontrols) and in all dose groups of females (low, mid and high dose: 19.2 g, 18.6 g and 15.4 gversus 23.5 g of controls). Urothelial hyperplasia was described to be mild in the high dosedfemales, and in males, thickening of the urothelial layer was minimal (detailed data were not given).Urinary bladder epithelial cell proliferation, characterized by labeling indices, increased withincreasing dosage of o-toluidine but was significant only in the 6000 ppm dosed males (1.42 versus0.47 of controls) and in the 6000 and 3000 ppm dosed females (2.55 and 0.38 versus 0.08 ofcontrols). Urinary excretion of NAAC was positively correlated with the cell proliferation in bothmale and female rats, according to the author, indicating that NAAC may be a useful urinarybiomonitor for o-toluidine exposure. Statistically significant and dose-related increase inmethemoglobin production was determined in all treated animals (males: 4.2 %, 10.7 %, and 14.9 %versus 0.6 % in controls; females: 6.2 %, 14.5 %, and 19.0 % versus 0.5 % in controls). Therefore aNOAEL could not be established. The LOAEL based on methemoglobinemia (male and females)and decreased body weight gain (females) was 500 ppm (adjusted to the test substance stability:app. 23.7 mg/kg bw/day for males and app. 25.5 mg/kg bw/day for females) (DuPont, 1994 cited by OECD/SIDS 2006)