Registration Dossier

Administrative data

Description of key information

According to OECD/SIDS of o-toluidine published by UNEP in 2006, repeated dose toxicity studies show that o-toluidine is markedly toxic to erythrocytes an a methemoglobin forming chemical. This was demonstrated in elevated methemoglobin levels up to 19.0 %  in the subacute feeding study as well vas marked splenic toxicity in the subacute gavage  and subchronic feeding studies, leading to hypercellularity in the bone marrow. Further target organs were liver and kidney (hemosiderin deposition) and urinary bladder (hyperplasia).  Based on the hematological findings no NOAEL could be derived, the LOAEL (rat, 14 -day feeding study) was 500 ppm (approx. 25.5 mg/kg bw/day for males and females, respectively)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
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
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
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
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
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
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).
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
Critical effects observed:
not specified
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)

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
25.5 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
There are studies available covering all relevant toxicological endpoints. This leads to the conclusion that o-toluidine is toxic to erythrocytes and a methemoglobin forming chemical. In addition there is evedence that o-toluidine is a mutagen and a carcinogen. o-toluidine is classified accordingly

Additional information

OECD / SIDS of o-toluidine reported subchronic feeding studies in which o-toluidine hydrochloride was administered to three groups of male Fisher 344 rats (n = 20) for 13 weeks (group one and two) and for 26 weeks (group three) at adosage of 5000 ppm. Following the 13 week feeding period group-two-animals were observed for13-weeks (recovery group) to determine the potential reversibility of the toxic effects (so-called stop-exposureexperiment).

The mean compound consumption, calculated from feed intake, was 301, 304 and 285 mg/kg bw/day, respectively (NCI, 1996). There were no treatment-related clinical signs of toxicity, no mortality; feed consumption was comparable in all groups. In the 13-week or 26-week continuous-exposure groups mean body weight gain was 21 % and 27 % lower for rats exposed to o-toluidine hydrochloride than those of the respective controls. At the end of the study the recovery groupshowed a slightly higher mean body weight gain than rats continuously exposed for 26 weeks (187versus 163 g). As target organs liver, spleen, kidneys were identified (for effects on testes: see the respective section). With regard to the liver, the relative liver weights were increased in the 13-week group (43 versus 35 g) and the 26-week group (46 versus 37 g), slight hemosiderin deposition was noted in 20/20 rats (versus 0/10 in controls) after 13 and 26 weeks which was partly reversible during the 13 week recovery as hemosiderin deposits were found only in 11/20 rats. When compared with controls, the right kidneys had absolute and relative increased weights (13 weeks:3.5 vs 3.27 g; 26 weeks: 4.0 vs 3.6 g) due to hemosiderin deposition in 20/20 rats of all three exposure groups (versus 0/10 in controls), which was reversible only with regard to the degree of hemosiderin deposition (mild to minimal). Urinary bladder epithelium showed mild hyperplasia in 10/20 and 17/18 rats (versus 0/10 in controls) after 13 and 26-week-treatment, respectively. These findings were not found after the 13-week recovery period. Spleen weights (relative and absolute) were significantly increased after every part of this investigation (rel. weights: 13 weeks:7.43 vs 2.12 g; 26 weeks: 9 vs 2.1 g; recovery: 3.23 vs. 2.08). The histopathological findings in alltreated rats included congestion, hematopoietic cell proliferation, and pigmentation due to hemosiderin deposition and fibrosis of the capsule, which increased in severity with duration ofexposure. Hematology was not performed (NCI, 1996).

According to OECD/SIDS of o-toluidine published by UNEP in 2006 o-toluidine was administered to 30 male F344 rats via gavage in a dose of 0 and 225 mg/kg bw/day for a total period of 20 days with interim kills after 5 and after 10 days of treatment. 4 rats died within the first 5 days of treatment and 6/30 rats were sacrificed on day 5,another 4 rats died up to the tenth day of treatment and 6 rats more were sacrificed at day 10. Within the final 10 days 2 rats died and the last 8 rats were sacrificed on day 20. Clinical signs were cyanosis, thinness and rough hair coat. Significantly reduced body weights was observed 5 and 10 days after treatment (p < 0.05) but not after 20 days of treatment (5, 10, 20 days, treated rats versus control rats: 183 g, 193 g, 226 g versus 214 g, 225 g, 233 g). Organs examined were liver, spleen, thyroid, urinary bladder and kidneys. Significantly (p < 0.005) increased spleen weights weredetermined at all time points: 5, 10, 20 days, treated rats versus control rats: 0.61 g, 1.10 g, 0.64 gversus 0.37 g, 0.42 g, 0.44 g. No differences were seen in weights of livers and kidneys when compared with the concurrent control rats. Mild renal hemosiderosis and mild tracheitis were observed (data not shown). Histopathological investigations showed at all time points significant splenic hemosiderosis (mean scores: 1.50, 2.33, 3.50 versus 0.0, 0.0, 0.30 of controls), congestion (2.67, 2.5, 2.5 versus 1.0, 1.3, 1.1 of controls) and hematopoesis (1.50, 3.17, 1.38 versus 0.0), which resulted most likely from methemoglobin formation which is supposed to occur due to the observed cyanosis. Hypercellularity in bone marrow was increased at all time points, significant only after 10 days of treatment (1.00, 2.33, 0.88 versus 0.0). Periacinar vascuolar degeneration was the only finding in the liver, which occurred also in the control rats (0.17, 1.33, 0.75 versus 1.60, 1.90, 0.30).No other organs were reported to be impaired. Hematology was not performed (Short 1983)

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 were observed in the high dose groups (males: 218 g versus 233 g of controls; females: 142 g versus 151 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 g versus 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 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 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 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 by OECD/SIDS 2006)

OVERALL CONCLUSION

According to OECD/SIDS of o-toluidine published by UNEP in 2006, repeated dose toxicity studies show that o-toluidine is markedly toxic to erythrocytes and a methemoglobin forming chemical. This was demonstrated in elevated methemoglobin levels up to 19.0 % in the subacute feeding study as well vas marked splenic toxicity in the subacute gavage and subchronic feeding studies, leading to hypercellularity in the bone marrow. Further target organs were liver and kidney (hemosiderin deposition) and urinary bladder (hyperplasia). Based on the hematological findings no NOAEL could be derived, the LOAEL (rat, 14 -day feeding study) was 500 ppm (approx. 25.5 mg/kg bw/day for males and females, respectively)


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
There are several publications - mainly 3 of them discussed in a woe approach - providing relevant and characteristic properties of o-toluidine although the reported studies do not comply totally with the requirements of today. They are therefore evaluated with Klimisch score 2

Justification for classification or non-classification

No classification is warranted because methemoglobinemia is an acute effect and taken into account for classification of acute effects.