N,N-dimethyl-p-toluidine

Endpoint summary

Description of key information

Repeated Dose toxicity- Oral

Repeated Dose toxicity- Inhalation

Repeated Dose toxicity- Dermal

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data is from peer reviewed journals

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

The toxicity potential of the test chemical was evaluated in a chronic oral toxicity study in rats.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: Rats were 5 to 7 weeks old at the beginning of the studies.
- Weight at study initiation:
- Fasting period before study:
- Housing: Male rats were housed up to three per cage, female rats were housed five per cage,
1. Cages : Polycarbonate (Lab Products, Inc., Seaford, DE), changed weekly (male mice) or twice weekly
2. Bedding: Irradiated Sani-Chips (P.J. Murphy Forest Products Corp., Montville, NY), changed weekly (male mice) or twice weekly
3. Cage Filters: Spun-bonded polyester (Snow Filtration Co., Cincinnati, OH), changed every 2 weeks
4. Racks: Stainless steel (Lab Products, Seaford, DE), changed and rotated every 2 weeks
- Diet (e.g. ad libitum): Irradiated NTP-2000 wafer feed (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly
- Water (e.g. ad libitum): Tap water (Columbus municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum
- Acclimation period: Animals were quarantined for 13 (male rats), 14 (female rats),

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72° ± 3° F
- Humidity (%): 50% ± 15%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

IN-LIFE DATES: From:Rats: October 20 (males) or 21 (females), 2004 To:Rats: October 17 (males) or 19 (females), 2006

Route of administration:

oral: gavage

Details on route of administration:

The chemical was administered by oral gavage because the chemical was not palatable by the feed rout

Vehicle:

corn oil

Details on oral exposure:

Details on oral exposure
PREPARATION OF DOSING SOLUTIONS: Dose formulations were prepared by adding the appro-priate amount of the test chemical to corn oil to achieve the desired concentration. Dose for-mulations were prepared three times for the 3-month studies.The 400 mg/mL dose formulation was prepared and observed to be a true solution, therefore, no homoge-neity or gavageability studies were performed. Stability studies of a 1.0 mg/mL formulation in corn oil were performed using GC/FID. Stability was confirmed for up to 44 days for formulations stored in amber glass containers sealed with Teflon®-lined lids, protected from light, at up to room temperature and for at least 3 hours under simulated animal room conditions.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test chemical dissolved in corn oil
- Concentration in vehicle: 0, 6, 20, or 60 mg/kg
- Amount of vehicle (if gavage): 2.5 mL/kg
- Lot/batch no. (if required): No data available
- Purity: No data available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Periodic analyses of the dose formulations were con-ducted by the study laboratory using GC/FID. During the 2-year studies, the dose formulations were analyzed at least every 3 months; animal room samples were also analyzed.Of the dose formulations analyzed and used, all 30 for rats were within 10% of the target concentrations; all 12 animal room samples for rats were within 10% of the target concentrations.

Duration of treatment / exposure:

2 year

Frequency of treatment:

5 days/week for 104 (male rats) or 105 weeks

Remarks:

0, 6, 20, or 60 mg/kg

No. of animals per sex per dose:

Groups of 50 male and 50 female rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on mortality in the 1,000 mg/kg groups, decreased (more than 10%) final mean body weights in the 125, 250, and 500 mg/kg male groups, and treatment-related nonneoplastic lesions in the liver, nose, spleen, kidney, and bone mar-row with increased severity at 125 mg/kg or greater, a high dose of 60 mg/kg test chemical was selected for the 2-year gavage study in rats. The low dose of 6 mg/kg was selected because this dose was reported to cause toxicity in humans (Potter et al., 1988). The doses selected for the 2-year gavage study in rats were 0, 6, 20, and 60 mg/kg, with a threefold dose spacing.
- Rationale for animal assignment (if not random): Animals were distributed randomly into groups of approximately equal initial mean body weights.
- Fasting period before blood sampling for clinical biochemistry:
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):
- Other:

Observations and examinations performed and frequency:

Observations and examinations performed & frequency
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked in table [No.?] were included: Survival was observed.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Daily, clinical findings were recorded every 4 weeks beginning with week 5 and at the end of the studies

BODY WEIGHT: Yes
- Time schedule for examinations: core study animals were weighed initially, weekly for the first 13 weeks, every 4 weeks thereafter, and at the end of the studies

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data available
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data available

FOOD EFFICIENCY:No data available
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data available
- Time schedule for examinations: No data available

OPHTHALMOSCOPIC EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available

HAEMATOLOGY: Yes / No / Not specified: yes
- Time schedule for collection of blood:Blood was collected from the retroorbital sinus of clinical pathology rats on day 86 for hematology.
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.: Hematology: hematocrit; hemoglobin and methemoglobin concentrations; erythrocyte, reticulocyte, platelet, and Heinz body counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials

CLINICAL CHEMISTRY: No data available
- Time schedule for collection of blood: No data available
- Animals fasted: No data available
- How many animals: No data available
- Parameters checked in table [No.?] were examined. No data available

URINALYSIS: No data available
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined. No data available

NEUROBEHAVIOURAL EXAMINATION: No data available
- Time schedule for examinations:No data available
- Dose groups that were examined: No data available
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data available

Sacrifice and pathology:

Sacrifice and pathology
Animals were sacrifice using Carbon dioxide asphyxiation

GROSS PATHOLOGY: Yes
Necropsies were performed on all core study animals.At necropsy, all organs and tissues were examined for grossly visible lesions, and all major tissues were fixed and pre-served in 10% neutral buffered formalin (except eyes were first fixed in Davidson’s solution), processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin for microscopic examination. For all paired organs (e.g., adrenal gland, kidney, ovary), samples from each organ were examined.

HISTOPATHOLOGY:
Complete histopathology was performed on all core study animals. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone (including marrow), brain, clitoral gland, esophagus, eye, gallbladder (mice), Harderian gland, heart (including aorta), large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), kidney, liver, lung (and mainstem bronchi), lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, uterus, and Zymbal’s gland (male rats).

Statistics:

Survival Analyses: Analysis of survival was estimated by the product-limit procedure of Kaplan and Meier . Statistical analyses for possible dose related effects on survival were analyzed by useing Cox’s method for testing two groups for equality and Tarone’s life table test to identify dose related trends. All reported P values for the survival analyses are two sided.Shirley and Dunn. Jonckheere’s test was used to analyszed by the significance of the dose related trends and to determine whether a trend sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose related trend (Dunnett’s or Dunn’s test).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings included signs of pallor in 60 mg/kg females and hyper-activity and boxing behavior in 20 mg/kg females and 60 mg/kg males and females. Hyperactivity and boxing behavior were first noticed during study month 8. All animals exhibited normal behavior prior to dosing. Boxing behavior, characterized by “kangaroo boxing” between cage mate pairs, was seen after dosing in 20 mg/kg females and 60 mg/kg males and females. In most months where this behavior was observed, the per-centage of animals displaying this behavior was greater following the first dose of the week compared to the percentage following the last dose of the week. The percentage of 60 mg/kg females displaying boxing behavior decreased over the course of the study.

Mortality:

mortality observed, treatment-related

Description (incidence):

Survival of 60 mg/kg males was significantly less than that of the vehicle controls. Although the survival of 60 mg/kg females was decreased compared to the vehicle controls, the decrease was not statistically significant

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of 60 mg/kg males were over 10% less than those of the vehicle controls after week 61 (day 421) and those of 60 mg/kg females were less than those of the vehicle controls after week 33 (day 225)

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The hematology findings in this 3-month interim evaluation were consistent with what occurred in the 3-month study. Increases in methemoglobin and Heinz bodies occurred in the 20 and 60 mg/kg male and female groups. Dose-related decreases occurred in the erythron characterized by decreases in hematocrit values, hemo-globin concentrations, and erythrocyte counts in the 20 and 60 mg/kg male and female groups. The erythron decreases were accompanied by trends toward erythrocyte macrocytosis and hypochro-mia evidenced by increases in the mean cell volume and decreases in the mean cell hemoglobin concentration values, respectively. Increases in reticulocyte counts demonstrated increased erythropoiesis in response to the decreased erythron. While the magnitudes of the eryth-ron decreases were not sufficient to categorically clas-sify these as anemias, the patterns of erythron changes were identical to what occurred in the 3-month study. At most, minimally decreased hemoglobin concentrations (decreased <5%), increased methemoglobin values (increased <20% in males only), and increased Heinz bodies (increased in females only) occurred in the 6 mg/kg groups.

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Administration of the test chemical resulted in increased incidences of nonneoplastic lesions of the liver and nasal cavity in male and female rats and the mesenteric lymph node in male rats and the kidney in male and female rats; the spleen and bone marrow in male and female rats

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There was clear evidence of carcinogenic activity of the test chemical in male F344/N rats based on increased incidences of hepatocellular carcinoma, and hepatocellular adenoma or carcinoma (combined), and increased incidences of nasal cavity neoplasms (primarily nasal cavity transitional epithe-lium adenoma). The increased incidences of thyroid gland follicular cell neoplasms may have been related to treatment. There was clear evidence of carcinogenic activity of the test chemical in female F344/N rats based on increased incidences of hepatocellular carcinoma and hepatocellular adenoma or carcinoma (combined). The occurrence of nasal cavity transitional epithelium adenoma was considered to be related to treatment.The occurrence of thyroid gland follicular cell neoplasms in male rats may have been related to treatment. The incidence of thyroid gland follicular cell adenoma was increased at the 60 mg/kg dose, and in addition, two thyroid gland follicular cell carcinomas occurred in the 60 mg/kg group. The incidence of follicular cell adenoma or carcinoma (combined) in the 60 mg/kg group exceeded the historical control ranges for corn oil gavage studies and for all routes of exposure.
The occurrence of nasal cavity transitional epithelium adenoma in female rats was considered to be related to treatment because these are rare neoplasms that have not occurred in the concurrent vehicle controls or in corn oil gavage historical controls and have occurred in only one of 1,196 historical control animals by all routes of exposure.The occurrence of thyroid gland follicular cell neoplasms in male rats may have been related to treatment. The incidence of thyroid gland follicular cell adenoma was increased at the 60 mg/kg dose, and in addition, two thyroid gland follicular cell carcinomas occurred in the 60 mg/kg group. The incidence of follicular cell adenoma or carcinoma (combined) in the 60 mg/kg group exceeded the historical control ranges for corn oil gavage studies and for all routes of exposure.

Other effects:

not specified

Dose descriptor:

LOAEL

Effect level:

6 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

histopathology: neoplastic

histopathology: non-neoplastic

mortality

Remarks on result:

other: toxic effects observed

Critical effects observed:

yes

Lowest effective dose / conc.:

6 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

kidney

liver

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Conclusions:

LOAEL was considered to be 6 mg/kg body weight /day when male and female rats were treated with test chemical orally.

Executive summary:

The toxicity potential of the test chemical was evaluated in a chronic oral toxicity study in rats.Groups of 50 male and 50 female Fischer 344 rats were administered 0, 6, 20, or 60 mg /kg body weight of test chemical in corn oil by gavage, 5 days per week for 104 or 105 weeks. Additional groups of 10 male and 10 female rats (clinical pathology study) were administered the same doses for 86 days. Survival of 60 mg/kg males was significantly less than that of the vehicle controls. Mean body weights of 60 mg/kg males and females were more than 10% less than those of the vehicle controls after week 61 and week 33, respectively. Clinical findings included signs of pallor in 60 mg/kg females and hyperactivity and boxing behavior in 20 mg/kg females and 60 mg/kg males and females. In the 20 and 60 mg/kg groups, there were dose-related decreases in hematocrit values, hemoglobin concentrations, and erythrocyte counts. There were similar trends toward erythrocyte macrocytosis and hypochromia and increased erythropoiesis as seen in the 3-month study. While the magni-tudes of the erythron decreases were not sufficient to classify the responses as anemias, the patterns of the erythron changes were identical to those in the 3-month study. In the liver of 60 mg/kg males and females, there were significantly increased incidences of hepatocellular carcinoma and hepatocellular adenoma or hepatocellular carcinoma (combined). Numerous nonneoplastic liver lesions occurred in dosed males and females primarily in the 20 and 60 mg/kg groups. In the nose, there were significantly increased incidences of transitional epithelium adenoma and transitional epithelium adenoma or carcinoma (combined) in 60 mg/kg males; transitional epithelium adenoma also occurred in female rats administered 6 or 60 mg/kg. In the nose, there were significantly increased incidences of nonneoplastic lesions in the olfactory, respiratory, and transitional epithelia of dosed rats. These lesions occurred with the greatest incidence and severity in the 60 mg/kg groups. The incidences of inflammation and nerve atrophy were significantly increased in males and females administered 60 mg/kg.There were increased incidences of follicular cell adenoma or carcinoma (combined) of the thyroid gland in all dosed groups of males, and an increased incidence of follicular cell adenoma in 20 mg/kg females.In the spleen, there were significantly increased incidences of hematopoietic cell proliferation in all dosed groups of males and females. The incidences of congestion and mesothelial hypertrophy of the capsule were significantly increased in 60 mg/kg males and all dosed groups of females. There were also significantly increased incidences of capsular fibrosis and atrophy of the lymphoid follicle in the 60 mg/kg groups. The incidences of pigmentation were significantly increased in all dosed groups of males and in 60 mg/kg females In all dosed groups of female rats, there were significantly increased incidences of nephropathy. Although the incidences of this lesion were not significantly increased in dosed males, the severities increased with increasing dose in both males and females. The incidences of pigmentation of the kidney were significantly increased in all dosed groups of males and in 60 mg/kg females.In the forestomach of males, there were significantly increased incidences of hyperplasia and ulcer in the 20 and 60 mg/kg groups and inflammation in the 60 mg/kg group. In the bone marrow of 20 and 60 mg/kg males and 60 mg/kg females, there were significantly increased incidences of hyperplasia. In the mesenteric lymph node of 20 and 60 mg/kg males, there were significantly increased incidences of histiocytic cellular infiltrates. Hence LOAEL was considered to be 6 mg/kg body weight /day when male and female rats were treated with test chemical orally.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

6 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Klimisch Rating 2

System:

hepatobiliary

Organ:

liver

lungs

nasal cavity

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Weight of evidence approach based on the various test chemicals

Justification for type of information:

Weight of evidence approach based on the various test chemicals

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Weight of evidence approach based on the various test chemicals

Principles of method if other than guideline:

Weight of evidence approach based on the various test chemicals

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: 2.Sprague-Dawley; 3. Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

no data available

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

3. GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Vapor atmospheres of the test chemical were generated under dynamic conditions using a gas bubbler (filling height ∼5 cm; diameter of glass flask ∼6 cm; content ≈100 g; maintained at 24 degrees C using a digitally controlled water bath from JULABO UC, Julabo, Seelbach, Germany).
- Method of holding animals in test chamber:
- Source and rate of air:
- Method of conditioning air:
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber: 24 degrees C
- Air flow rate: The low and intermediate concentrations were generated by airflows of 1.4 and 8.0 L/min through the bubbler while maintaining a total air flow rate through the inhalation chamber of 15 L/min.
- Air change rate:
- Method of particle size determination:
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used: GC
- Samples taken from breathing zone: yes/no: yes

VEHICLE (if applicable)
- Justification for use and choice of vehicle:
- Composition of vehicle:
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle:
- Lot/batch no. of vehicle (if required):
- Purity of vehicle:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

3. Exposure atmospheres were characterized using a gas chromatographic (GC) technique (Hewlett-Packard GC 5880A equipped with a flame ionization detector [FID] and fused silica columns, length 10 m, ID 0.53 mm, dimethylpolysiloxane film thickness 2.7μm). For GC analyses, samples were taken from the vicinity of the rats’ breathing zone three times per exposure day using two adsorption tubes (in-line) filled with the chromatographic adsorbent Florisil (Merck, Darmstadt, Germany, article no. 12518, 60–100 mesh), which samples both the vapor and aerosol phase with a high collection efficiency. Adsorption
tubes were eluted with ethanol. The collection efficiency was determined from adsorption tubes spiked with the test chemical and subsequently subjected to an airflowsimilar to that used for the sampling of atmospheres. The recovery was 98.3% and the coefficient of variation was 2.3%. No breakthrough into the second adsorption tube was observed in any of the samples. The test atmospherewas withdrawn from the inhalation chamber using a calibrated gas metering device at a flowrate of 1.0 L/min, and sampling durations ranged from 10 to 20 min. The temporal stability of test atmospheres was monitored continuously using a total hydrocarbon analyzer (Compur, Munich, Germany) and a RAS-2 aerosol photometer (MIE, Bedford, MA) for the vapor and aerosol exposure atmospheres, respectively. The relative humidity in inhalation chambers was in the range of 7–10% in the vapor exposure groups and 43% in the aerosol exposure group.

Duration of treatment / exposure:

2. 2 weeks
3.

Frequency of treatment:

2. Frequency of treatment: 6 hr/day, 5 days/week
Post. obs. period: 2 weeks, control and high exposure groups
3. 6 h/day, 3–5 days/wk, 20–22 exposures in total

Remarks:

Study 2- 0, 5.6, 32.8, 67.6 ppm

Remarks:

Study 3: Exposure was to the following mean analytical concentrations:
0 (dry air), 19.1, 115.1, and 702.3 mg/m3

No. of animals per sex per dose:

3. 10 rats/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

3. - Dose selection rationale:
Prior to the 4-wk study, a 5 × 6-h pilot study was performed to validate technical procedures and to select appropriate exposure concentrations. Five groups of rats, each consisting
of 10 rats/sex/group, were exposed to 0 (dry air), 27.1, 104.8, 381.6, and 1283.7 mg/m3 air. Shortly after the fifth exposure, 5 rats/sex/group were bled via venipuncture from the orbital sinus for hematological determinations, while the blood for clinical pathology was collected by heart puncture during necropsy on the first postexposure day. The weights of the following organs were determined: brain, liver, lung, heart, kidneys, and spleen. The remaining 5 rats/sex/group were necropsied (gross necropsy only) after a 2-wk post-exposure recovery period. Based on the minimal hematotoxicity observed at 381.6 mg/m3 in female rats, the no-observed-adverse effect level (NOAEL) of this 1-wk pilot study was 105 mg/m3.

Observations and examinations performed and frequency:

2. CAGE SIDE OBSERVATIONS: Yes / No / Not specified : yes
- Time schedule:
- Cage side observations checked in table [No.?] were included. : mortality

DETAILED CLINICAL OBSERVATIONS: Yes / No / Not specified : yes
- Time schedule:

BODY WEIGHT: Yes / No / Not specified L: yes
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes / No / Not specified
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes / No / Not specified : yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes / No / Not specified : yes
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes / No / Not specified
- Time schedule for analysis:
- Dose groups that were examined:
- Number of animals:
- Parameters checked in table [number] were examined.
LUNG BURDEN: Yes / No / Not specified
- Time schedule for analysis:
- Dose groups that were examined:
- Number of animals:
- Parameters checked in table [number] were examined.

OTHER:
3. CAGE SIDE OBSERVATIONS: Yes / No / Not specified
: yes
- Time schedule:
During the study, animals were observed before and after each exposure for signs of toxicity.
- Cage side observations checked in table [No.?] were included.
: During the study, animals were observed before and after each exposure for signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes / No / Not specified
: yes
- Time schedule: During the study, animals were observed before and after each exposure for signs of toxicity.

BODY WEIGHT: Yes / No / Not specified
: yes
- Time schedule for examinations: Body weights were recorded twice/week.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes / No / Not specified
: yes
- Time schedule for examinations: Ophthalmic exams were performed on 5 animals/sex/group before initiation of dosing and
prior to termination
- Dose groups that were examined:

HAEMATOLOGY: Yes / No / Not specified
: yes
- Time schedule for collection of blood:
Blood was collected retro-orbitally at study midterm in 5 rats/sex/group for selected hematology and clinical pathology endpoints
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
: no data available
- How many animals:

- Parameters checked in table [No.?] were examined.
: The following hematology parameters were evaluated in all rats at terminal sacrifice: red blood cell count, red blood cell morphology, hematocrit,
hemoglobin, mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), mean cell volume (MCV), leukocyte count, reticulocyte counts, Heinz bodies, platelet (thrombocyte) count, prothrombin time.

CLINICAL CHEMISTRY: Yes / No / Not specified
: yes
- Time schedule for collection of blood: Blood was collected retro-orbitally at study midterm in 5 rats/sex/group for selected hematology and clinical pathology endpoints
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.
: The following serum clinical chemistry parameters were evaluated in all rats: alanine aminotransferase (ALAT), albumin, alkaline phosphatase, aspartate aminotransferase (ASAT), blood urea nitrogen, cholesterol, creatine kinase, creatinine, glucose, glutamate dehydrogenase (GLDH), lactate dehydrogenase (LDH), leucine arylamidase (LAP), serum protein electrophoresis, total bilirubin, total protein, triglycerides, and the electrolytes calcium, inorganic phosphorus, magnesium, sodium, potassium, and chloride. In hepatic tissue, cytochrome P-450, N-demethylase, O-demethylase, and triglycerides were determined.

URINALYSIS: Yes / No / Not specified
: yes
- Time schedule for collection of urine: Overnight urine was sampled toward the end of the study
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.
: sediment composition, osmolality, pH, protein, glucose, blood, bilirubin, urobilinogen, and ketone bodies

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes / No / Not specified
- Time schedule for analysis:
- Dose groups that were examined:
- Number of animals:
- Parameters checked in table [number] were examined.
LUNG BURDEN: Yes / No / Not specified
- Time schedule for analysis:
- Dose groups that were examined:
- Number of animals:
- Parameters checked in table [number] were examined.

OTHER:

Sacrifice and pathology:

2. GROSS PATHOLOGY: Yes (see table) / No / No data: yes
HISTOPATHOLOGY: Yes (see table) / No / No data: yes
3. GROSS PATHOLOGY: Yes (see table) / No / No data: yes, All animals of the 4-wk study were sacrificed by exsanguination (heart puncture) under diethyl ether anesthesia. Organ weights were recorded for adrenals, brain, heart, kidneys, liver, lungs, ovaries, spleen, testes, thymus, and thyroid. Organs and tissues were preserved in 10% neutral-buffered formalin. Prior to trimming, the head was decalcified in formic acid.The organs/tissues were embedded in paraffin, cut (approximately 5μm), and stained with hematoxylin and eosin (H&E). The nasal cavitywas transversely sectioned at four levels largely consistent with the procedure described byYoung (1981) and the larynx was sectioned at the pouch level according to Lewis (1981).
HISTOPATHOLOGY: Yes (see table) / No / No data: yes, Microscopic examinations of H&E sections of the following tissues were performed for all animals: adrenals, aorta (thoracic), bone (femur), bone marrow (sternum and femur), brain (cerebrum, cerebellum, and medulla oblongata), coagulating glands,colon, cecum, rectum, duodenum, jejunum, ileum, esophagus,exorbital lacrimal glands, eyes and eyelids, harderian glands, heart, joint (knee), kidneys (including renal pelvis), lachrymal glands, larynx, liver, lungs (intratracheally instilled with buffered formaldehyde using pressure of 20 cm H2O), mammary glands (females), lymph nodes (mandibular, mesenteric, lung-associated), nasopharyngeal tissues, ovaries, pancreas, pituitary, prostate, mandibular salivary gland, seminal vesicles, sciatic nerve, skeletal muscle (thigh), skin, spinal cord (cervical, midthoracic, and lumbar), spleen, stomach, thymus, thyroid(including parathyroids), tongue, trachea, urinary bladder (instilled),uterus with cervix and vagina, testis, and epididymis including vas deferens. A special stain for the visualization of lipids was utilized on cryo-cut liver sections (oil red O). The degree of hemosiderosiswas examined using Prussian blue-stained sections of liver and spleen. Bone-marrow smears (from femur) were stained with May–Giemsa–Grunwald, and for differentiation 1000 cells/smear were counted by light microscopy.

Other examinations:

no data available

Statistics:

3.Body weights, hematology, and clinical pathology data were compared by the Wilcoxon U-test. Bone-marrow cell counts, reticulocyte counts, methemoglobin, and organ weight data were analyzed using a one-way analysis of variance (ANOVA) and Tukey–Kramer post hoc test. Prior to analysis of the reticulocyte and Heinz body counts, data were arcsine square root transformed. Pairwise comparisons of prebled and non-prebled rats within a group used a Holm–Sidak test of arcsine square root transformed data. This test and the Pearson Product Moment Correlation test was calculated using SigmaStat 3.1 for Windows (Systat Software, Inc., Point Richmond, CA). Histopathological findings were compared with the concurrent control using Fisher’s exact test. For all tests the criterion for statistical significance was set at p < .05.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

3. Exposure up to 115.1 mg/m3 was tolerated without clinical signs and mortality. Rats exposed to 702.3 mg/m3 displayed signs, most of them related to a cyanotic appearance, respiratory distress and breathing sounds, bradypnea, nasal discharge and irritation, and reduced motility.

Mortality:

no mortality observed

Description (incidence):

2. There were no deaths observed
3. At 702.4 mg/m3, 1 of 10 female rats succumbed on study day 7 (first exposure was on day 0).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

2. There were no changes in body weights
3. Body weights were significantly decreased

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

2. There were no changes in food consumption

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

3. There was no evidence of test compound-related ocular effects.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

2. Methemoglobinemia was significantly increased across all exposure groups at both terminal and recovery necropsies in both sexes.
3. Hematological parameters were significantly affected at 702.3 mg/m3, and changes were characterized by decreased hemoglobin, red blood cell counts, MCHC, and hematocrit, and increased MCH, MCV, methemoglobin, reticulocyte counts and erythrocytes containing Heinz bodies. Red blood cells exibited polychromasia and a somewhat equivocal poikilocytosis. The increased mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) and decreased mean corpuscular hemoglobin concentration (MCHC), which indicate that the anemia was macrocytic and hyperchromic, may have been confounded by the presence of large numbers of Heinz bodies and reticulocytosis. Conclusive changes in the total leukocyte counts did not occur whereas leukocyte differentials revealed a decreased percentage of lymphocytes and an increased percentage of segmented leukocytes . Increased counts of erythrocytes containing Heinz bodies occurred at 115.1 mg/m3 and above. Thrombocyte counts were indistinguishable between the groups, while the prothrombin time was increased at 702.3 mg/m3

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

3. Statistical analysis of data revealed in serum significantly increased alkaline phosphatase and leucine aryl amidase activities, increased concentrations of total bilirubin, and decreased cholesterol at 702.3 mg/m3. Some statistically significant effects (cholesterol) of unclear pathognostic significance were also found at 115.1 mg/m3 and above. The electrolytes magnesium and inorganic phosphorous were statistically significantly increased in females at 702.3 mg/m3. At the high exposure concentration, serum protein electrophoresis demonstrated changes in the albumin:globulin fractions. Mild changes of hepatic monooxygenase activities and triglycerides were noticed at this exposure level, although a clear concentration dependence did not occur.

Urinalysis findings:

no effects observed

Description (incidence and severity):

3. Urinalysis did not exhibit any consistent differences among the groups

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

3. Organ weight changes included a significant increase of absolute and relative weights of spleen (male and females) and liver (females only) at 702.3 mg/m3. Thymus weights were decreased at 115.1 mg/m3 and above. Some slight elevations of the kidney to body weight ratio occurred in the females of the high exposure group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

2. Enlarged spleens, increased production of red cells in the spleen, bone marrow, and liver, and other hematology changes were consistent with induction of hemolytic anemia. Kidney effects were considered secondary to hemolytic anemia. This condition was not completely reversed at the end of the 14 day recovery period.
3. Microscopic examination of the rats revealed changes in the spleen at 115.1 mg/m3 (females only) and at 702.3 mg/m3 (males and females)

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

2. The increase in methemoglobin was not accompanied by adverse histopathology or clinical signs.
3. Lesions indicative of specific splenic or hepatic tissue damage were not observed. In the nasal cavities an atrophy of the olfactory epithelium occurred at 115.1 mg/m3 and above without evidence of lesions of the respiratory epithelium. In the proximal nasal cavity two female rats exposed to 702.3 mg/m3 displayed an epithelial hyperplasia, hyperkeratosis, and necrotic changes. Specific lesions attributable to the exposure of this test chemical were not found in the remaining tissues examined, including the entire respiratory tract. The most salient finding of this quantitative analysis was consistent with an elevated hematopoiesis, especially in the female rats of the high-level exposure group, as indicated by the increased counts of normoblasts , the precursor cells of reticulocytes. Adverse effects on specific cell populations of the bone marrow were not found at any concentration, and the changes observed were typical of a compensatory increase in erythropoiesis

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Dose descriptor:

NOAEC

Effect level:

5.6 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

Critical effects observed:

not specified

Conclusions:

Based on the available results and applying the weight of evidence approach, the NOAEC can be considered to be 19.1 mg/m3

Executive summary:

Various studies have been reviewed to evaluate the toxicity potential of the test chemical when exposed repeatedly via inhalation route. These include in vivo experimental studies performed on rats for the test chemical. The results are mentioned below:

A study was performed according to OECD 412 to evaluate the toxicity potential of the test chemical when exposed to rats via inhalation route. Sprague Dawley rats were exposed to 5.6, 32.8, 67.6 ppm of the test chemical 6 hr/day, 5 days/week for 2 weeks with post exposure period of 2 weeks, control and high exposure groups. .There were no deaths, and no changes in body weights, food consumption, clinical observations, or clinical chemistry. Methemoglobinemia was significantly increased across all exposure groups at both terminal and recovery necropsies in both sexes. Enlarged spleens, increased production of red cells in the spleen, bone marrow, and liver, and other hematology changes were consistent with induction of hemolytic anemia. Kidney effects were considered secondary to hemolytic anemia. This condition was not completely reversed at the end of the 14 day recovery period. A "no effect level" was not established under the conditions of this study. However, the lowest exposure level of 5.6 ppm was considered a "no adverse effect level" because the increase in methemoglobin was not accompanied by adverse histopathology or clinical signs.

This result is supported by a study whose objective and focus of the toxicity was to evaluate the toxicity by a 4-wk inhalation exposure to the test chemical. The study was performed according to OECD 412 Guidelines.Groups of 10 rats/sex were nose-only exposed to mean analytical concentrations of 19.1, 115.1, and 702.3 mg/m3 using an exposure regimen of 6 h/day and 20–22 exposures within a time period of 4 wk. These concentrations were selected based on results from a repeated 5×6 h/day pilot study using concentrations of 27.1, 104.8, 381.6, and 1283.7 mg/m3. Animals subjected to necropsy were sacrificed 1 day after the last exposure. During the study, animals were observed before and after each exposure for signs of toxicity. Body weights were recorded twice/week. Rectal temperatures were measured shortly after exposure using a rectal probe repetitively during the course of study.Ophthalmic exams were performed on 5 animals/sex/group before initiation of dosing and prior to termination. Blood was collected retro-orbitally at study midterm in 5 rats/sex/group for selected hematology and clinical pathology endpoints. The following serum clinical chemistry parameters were evaluated in all rats: alanine aminotransferase (ALAT), albumin, alkaline phosphatase, aspartate aminotransferase (ASAT), blood urea nitrogen, cholesterol, creatine kinase, creatinine, glucose, glutamate dehydrogenase (GLDH), lactate dehydrogenase (LDH), leucine arylamidase (LAP), serum protein electrophoresis, total bilirubin, total protein, triglycerides, and the electrolytes calcium, inorganic phosphorus, magnesium, sodium, potassium, and chloride. In hepatic tissue, cytochrome P-450, N-demethylase, O-demethylase, and triglycerides were determined. All animals of the 4-wk study were sacrificed by exsanguination (heart puncture) under diethyl ether anesthesia. Organ weights were recorded for adrenals, brain, heart, kidneys, liver, lungs, ovaries, spleen, testes, thymus, and thyroid. Organs and tissues were preserved in 10% neutral-buffered formalin. Prior to trimming, the head was decalcified in formic acid.The organs/tissues were embedded in paraffin, cut (approximately 5μm), and stained with hematoxylin and eosin (H&E). The nasal cavity was transversely sectioned at four levels largely consistent with the procedure described byYoung (1981) and the larynx was sectioned at the pouch level according to Lewis (1981). The following hematology parameters were evaluated in all rats at terminal sacrifice: red blood cell count, red blood cell morphology, hematocrit, hemoglobin, mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), mean cell volume (MCV), leukocyte count, reticulocyte counts, Heinz bodies, platelet (thrombocyte) count, prothrombin time. Exposure up to 115.1 mg/m3 was tolerated without clinical signs and mortality. Rats exposed to 702.3 mg/m3 displayed signs, most of them related to a cyanotic appearance, respiratory distress and breathing sounds, bradypnea, nasal discharge and irritation, and reduced motility.Rectal temperatures were significantly decreased at 115.1 mg/m3 and above. Dark and enlarged spleens occurred at 702.3 mg/m3. At this concentration, prominent treatment-related effects included methemoglobinemia, reticulocytosis, red blood cells with Heinz bodies, decreased hemoglobin, hematocrit, and red blood cell counts. Borderline evidence of erythrocytotoxicty was noticed at 115.1 mg/m3 (based on a minimal increase in Heinz bodies). Spleen and liver weights were significantly increased at 702.3 mg/m3, whereas the thymus weight was decreased at 115.1 mg/m3 and above. Microscopic changes were found in the spleen (hemosiderosis) at 702.3mg/m3.Anatrophy of the olfactory epithelium in the nasal cavities occurred at 115.1mg/m3 and above. Clinical pathology revealed changes pathognostic of hepatic effects, although microscopic examinations did not reveal any specific changes. The no-observed-adverse-effect level (NOAEL) of the 4-wk study was 19.1 mg/m3 and is based on the predominant atrophic changes of the olfactory epithelium and the minimal to borderline erythrocytotoxic effects at 115.1 mg/m3.

Based on the available results and applying the weight of evidence approach, the NOAEC can be considered to be 19.1 mg/m3

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

19.1 mg/m³

Study duration:

subacute

Experimental exposure time per week (hours/week):

6

Species:

rat

Quality of whole database:

Klimisch Rating 2

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

waiver

Additional information

Repeated Dose Oral

Various studies have been reviewed to evaluate the toxicity of the test chemical when dosed repeatedly via oral route. These include in vivo experimental studies performed rats as well as mice for the test chemical. The results are mentioned below:

The toxicity potential of the test chemical was evaluated in a chronic oral toxicity study in rats.Groups of 50 male and 50 female Fischer 344 rats were administered 0, 6, 20, or 60 mg /kg body weight of test chemical in corn oil by gavage, 5 days per week for 104 or 105 weeks. Additional groups of 10 male and 10 female rats (clinical pathology study) were administered the same doses for 86 days. Survival of 60 mg/kg males was significantly less than that of the vehicle controls. Mean body weights of 60 mg/kg males and females were more than 10% less than those of the vehicle controls after week 61 and week 33, respectively. Clinical findings included signs of pallor in 60 mg/kg females and hyperactivity and boxing behavior in 20 mg/kg females and 60 mg/kg males and females. In the 20 and 60 mg/kg groups, there were dose-related decreases in hematocrit values, hemoglobin concentrations, and erythrocyte counts. There were similar trends toward erythrocyte macrocytosis and hypochromia and increased erythropoiesis as seen in the 3-month study. While the magni-tudes of the erythron decreases were not sufficient to classify the responses as anemias, the patterns of the erythron changes were identical to those in the 3-month study. In the liver of 60 mg/kg males and females, there were significantly increased incidences of hepatocellular carcinoma and hepatocellular adenoma or hepatocellular carcinoma (combined). Numerous nonneoplastic liver lesions occurred in dosed males and females primarily in the 20 and 60 mg/kg groups. In the nose, there were significantly increased incidences of transitional epithelium adenoma and transitional epithelium adenoma or carcinoma (combined) in 60 mg/kg males; transitional epithelium adenoma also occurred in female rats administered 6 or 60 mg/kg. In the nose, there were significantly increased incidences of nonneoplastic lesions in the olfactory, respiratory, and transitional epithelia of dosed rats. These lesions occurred with the greatest incidence and severity in the 60 mg/kg groups. The incidences of inflammation and nerve atrophy were significantly increased in males and females administered 60 mg/kg.There were increased incidences of follicular cell adenoma or carcinoma (combined) of the thyroid gland in all dosed groups of males, and an increased incidence of follicular cell adenoma in 20 mg/kg females.In the spleen, there were significantly increased incidences of hematopoietic cell proliferation in all dosed groups of males and females. The incidences of congestion and mesothelial hypertrophy of the capsule were significantly increased in 60 mg/kg males and all dosed groups of females. There were also significantly increased incidences of capsular fibrosis and atrophy of the lymphoid follicle in the 60 mg/kg groups. The incidences of pigmentation were significantly increased in all dosed groups of males and in 60 mg/kg females In all dosed groups of female rats, there were significantly increased incidences of nephropathy. Although the incidences of this lesion were not significantly increased in dosed males, the severities increased with increasing dose in both males and females. The incidences of pigmentation of the kidney were significantly increased in all dosed groups of males and in 60 mg/kg females.In the forestomach of males, there were significantly increased incidences of hyperplasia and ulcer in the 20 and 60 mg/kg groups and inflammation in the 60 mg/kg group. In the bone marrow of 20 and 60 mg/kg males and 60 mg/kg females, there were significantly increased incidences of hyperplasia. In the mesenteric lymph node of 20 and 60 mg/kg males, there were significantly increased incidences of histiocytic cellular infiltrates. Hence LOAEL was considered to be 6 mg/kg body weight /day when male and female rats were treated with test chemical orally.

This result is supported by a similar chronic oral toxicity study performed on mice. Groups of 50 male and 50 female B6C3F1 mice were administered 0, 6, 20, or 60 mg/kg body weight of the test chemical in corn oil by gavage, 5 days per week for 105 weeks. Survival of 60 mg/kg females was significantly less than that of the vehicle control group. Mean body weights of 60 mg/kg males and females were more than 10% less than those of the vehicle controls after week 89 and week 65, respectively. In the liver, there were significantly increased incidences of hepatocellular adenoma in 20 and 60 mg/kg females and hepatocellular carcinoma in 60 mg/kg males and all dosed female groups. The increased incidences of hepatocellular adenoma and carcinoma in the dosed groups were primarily due to increased incidences of animals with multiple hepatocellular neoplasms. The incidences of hepatoblastoma were significantly increased in 20 mg/kg males and 60 mg/kg males and females. In all dosed groups of males and females, there were significantly increased incidences of hepatocyte hypertrophy, and the incidences of eosinophilic focus were significantly increased in the 20 and 60 mg/kg males and females. There were significantly increased incidences of fatty change and necrosis in 60 mg/kg females. In the lung of 20 and 60 mg/kg female mice, there were significantly increased incidences of alveolar/ bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined). There were also significantly increased incidences of alveolar epithelium hyperplasia in 20 mg/kg females; bronchiolar epithelium regeneration, bronchus epithelium regeneration, and bronchus necrosis in 60 mg/kg females; and alveolar infiltrates of histiocytes in 60 mg/kg males and females. In the forestomach of 20 and 60 mg/kg female mice, there were significantly increased incidences of squamous cell papilloma and squamous cell papilloma or carcinoma (combined). There were significantly increased incidences of epithelium hyperplasia in 20 and 60 mg/kg females, and inflammation and ulcer in 60 mg/kg females. In the nose, there were significantly increased incidences of nonneoplastic lesions of the olfactory and respiratory epithelia in 60 mg/kg males and 20 and 60 mg/kg females. There were significantly increased incidences of nerve atrophy in 60 mg/kg males and females and of inflammation in 60 mg/kg females. The incidences of atrophy of the olfactory lobe were significantly increased in 60 mg/kg males and females. In 60 mg/kg females, the incidences of regenerative hyperplasia of the nasolacrimal duct and necrosis of the vomeronasal organ were significantly increased. In the spleen, there was a significantly increased incidence of atrophy in 60 mg/kg females. There were also significantly increased incidences of bone marrow hyperplasia in all dosed groups of females and mesenteric lymph node atrophy in 60 mg/kg females. Hence, LOAEL was considered to be 20 mg/kg body weight /day when male and female rats were treated with test chemical orally.

These results are also supported by another chronic oral toxicity study performed on rats. For the chronic studies, male and female F344/N rats (50 animals/sex/dose) received the test chemical for up to 104 weeks at doses of 0, 6, 20, or 60 mg/kg. Because of the hematologic toxicity and body weights 10% lower than controls at doses of greater than 60 mg/kg in the subchronic studies, the high dose selected for the chronic study was 60 mg/kg in rats. Complete necropsies were performed on all animals after sacrifice when moribund or at the end of the 2-year exposure period. At necropsy, all organs and tissues were examined for grossly visible lesions. Tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The following tissues were examined microscopically from male and/or female animals: gross lesions and tissue masses, adrenal gland, bone with marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary glands, nose, ovary, pancreas, pancreatic islets, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder,and uterus. Following completion of the studies, the accuracy of the histopathologic diagnoses was determined by NTP pathology reviews. The test chemical induced toxic and/or carcinogenic effects in the hematologic system, liver, nasal cavity, and other organ systems in rats. The hematologic toxicity was characterized by an increase in methemoglobin levels and a macrocytic, hypochromic, responsive anemia particularly in rats. The anemia was defined as an insufficient concentration of hemoglobin and erythrocytes larger than their normal volume. The mechanism for the anemia was thought to involve oxidative damage to hemoglobin leading to Heinz body formation and decreased erythrocyte survival. Based on these effects the LOAEL value for the chronic study can be considered to be 20 mg/kg/day.

In a similar chronic oral toxicity study, groups of 50 male and female B6C3F1 mice were used for the study. The test chemical was prepared for oral gavage administration in corn oil to deliver the target concentrations of the chemical (0, 6, 20 or 60 mg/kg) to mice in a volume of 5 ml/kg body weight. Because of the hematologic toxicity and body weights 10% lower than controls at doses of greater than 60 mg/kg in the sub-chronic studies, the high dose selected for the chronic study was 60 mg/kg in mice. For the chronic studies, male and female B6C3F1 mice received 0, 6, 20 or 60 mg/kg of the test chemical in corn oil upto 104 weeks. Complete necropsies were performed on all animals after sacrifice when moribund or at the end of the 2-year exposure period. At necropsy, all organs and tissues were examined for grossly visible lesions. There was decreased survival, particularly in high-dose females, that was considered to be related to tumor formation. The final mean body weights of high-dose mice were lower than controls. In male mice, there was an increase in multiple hepatocellular adenomas at the top 2 dose levels, an increase in hepatocellular carcinomas at the top dose level, and an increase in hepatoblastomas at the top 2 dose levels. The combined incidence of hepatocellular adenomas or carcinomas or hepatocellular adenomas, and of hepatocellular carcinomas and hepatoblastomas (combined) was also significant. In female mice, there was an increase in the incidence of hepatocellular adenomas at the top 2 dose levels, an increase in the total incidence of hepatocellular carcinomas (total including multiple) at all 3 dose levels, and an increase in hepatoblastomas at the top dose levels. The combined incidence of hepatocellular adenomas or carcinomas or hepatocellular adenoma, hepatocellular carcinomas and hepatoblastomas (combined) at the top 2 dose levels was also significant. Hepatocellular hypertrophy was seen at the 2 highest dose levels in rats and in all 3 dose levels in mice. In addition, there were increases in non-neoplastic forestomach lesions in female mice including epithelium hyperplasia.Based on these observations, the LOAEL value can be considered to be 6 mg/kg.

Furthermore, the above chronic studies are supported by a sub-chronic oral toxicity study performed on rats. Groups of 10 male and 10 female Fischer 344 rats were administered 0, 62.5, 125, 250, 500, or 1,000 mg /kg body weight of test chemical in corn oil by gavage, 5 days per week for 14 weeks. Additional groups of 10 male and 10 female rats (clinical pathology study) were administered the same doses, 5 days per week for 25 days. On day 88, blood was collected from core study rats for hemoglobin and methemoglobin analyses only. All 1,000 mg/kg male and female rats and one 500 mg/kg male rat died by study day 3. Mean body weights of all surviving dosed groups of males and females were significantly less than those of the vehicle controls. Clinical findings associated with exposure to test chemical included cyanosis, abnormal breathing, and lethargy in groups administered 250 mg/kg or greater. Methemoglobinemia appeared to be the primary hematologic toxic response, and many other lesions could be explained as secondary to methemoglobin formation including Heinz body formation; a macrocytic, hypo-chromic, responsive anemia; and increased hematopoietic cell proliferation in the spleen and bone marrow. In general, hematologic changes were dose-related and occurred at both evaluated timepoints in all dosed groups. Anemia was evidenced by decreases in hematocrit values, hemoglobin concentrations, and erythrocyte counts; erythrocyte macrocytosis was characterized by increases in mean cell volume and mean cell hemoglobin values; erythrocyte hypochromia was evidenced by decreases in mean cell hemoglobin concentration values; and an erythropoietic response to the anemia was characterized by substantially increased reticulocyte and nucleated erythrocyte counts. Liver weights of all surviving dosed groups of males and females were significantly greater than those of the vehicle controls. Kidney weights of all surviving dosed groups of females were significantly greater than those of the vehicle controls. There were significant decreases in left cauda epididymis and left epididymis weights in 250 mg/kg males. In the surviving groups of rats, there were significantly increased incidences of pigmentation in the liver of all dosed groups, hepatocyte hypertrophy in groups administered 125 mg/kg or greater, and hepatocyte necrosis in 62.5, 250, and 500 mg/kg females. In the olfactory epithelium of the nose, there were dose related increases in the incidences and severities of degeneration in all dosed groups and significantly increased incidences of metaplasia in the 250 and 500 mg/kg groups. In the respiratory epithelium of the nose, there were significantly increased incidences of hyperplasia and squamous metaplasia in all of the groups administered 125 mg/kg or greater. The incidences of glandular hyperplasia of the nose were significantly increased in males and females administered 125, 250, or 500 mg/kg. In the spleen, there were significantly increased incidences of capsule fibrosis, congestion, mesothelial hypertrophy, and lymphoid follicle atrophy primarily in groups administered 125 mg/kg or greater. Hematopoietic cell proliferation and pigmentation were increased in severity in treated groups. In the kidney, there were significantly increased incidences of nephropathy (females), pigmentation (males and females), papillary necrosis (males and females), and mineralization (males). Other treatment-related lesions included inflammation of the forestomach in males, mesenteric lymph node atrophy in females, and bone marrow hyperplasia in males and females. Low Observed Adverse Effect Level (LOAEL) was considered to be 62.5 mg/kg/day, when male and female rats were treated with test chemical orally.

Additionally in another comparable sub-chronic oral toxicity study in mice, groups of 10 male and 10 female B6C3F1 mice were administered 0, 15, 30, 60, 125, or 250 mg /kg body weight in corn oil by gavage, 5 days per week for 14 weeks. All 250 mg/kg male and female mice (except for one male mouse) died before day 10, and three males and two females administered 125 mg/kg died before the end of the study. The final mean body weight of 125 mg/kg males and the mean body weight gains of 125 mg/kg males and females were significantly less than those of the vehicle controls. Clinical findings associated with administration of test chemical included abnormal breathing, thinness, lethargy, cyanosis, and ruffled fur in 125 and 250 mg/kg males and females. Methemoglobinemia appeared to be the primary hematologic toxic response; however there were less severe erythron changes compared to the 3-month study in rats. In females, no erythron changes were detected up to 125 mg/kg. In males, inconsistent and minor decreases in hematocrit values, hemoglobin concentrations, and erythrocyte counts, and increased reticulocyte counts occurred in groups administered 60 mg/kg or greater. Methemoglobin values were minimally increased in males and females administered 30 mg/kg or greater. Heinz bodies were slightly increased in 60 mg/kg females, 125 mg/kg males and females, and the one surviving 250 mg/kg male; Heinz body formation was considered secondary to methemoglobin formation. Liver weights of all dosed groups of mice were significantly greater than those of the vehicle controls. There were no changes in the proportion of regularly cycling females, estrous cycle length, or percentage of time spent in the individual stages of the estrous cycle of female mice at any dose. There were no significant differences in any of the reproductive organ weights or sperm parameters of male mice at any dose. In the surviving groups of mice, there were significantly increased incidences of bronchiolar epithelium degeneration, bronchiolar epithelium regeneration, and peri-bronchiolar chronic active inflammation in the lung of 125 mg/kg groups, and histiocytic infiltrates of the alveoli in 125 mg/kg females. In the nose, there were significantly increased incidences of glandular hyperplasia and olfactory epithelium metaplasia in the 125 mg/kg groups and olfactory epithelium degeneration in 60 mg/kg females and 125 mg/kg males and females. In the thymus, the incidences of thymocyte necrosis in the 125 mg/kg groups were significantly increased. In the liver, the severities of cytoplasmic vacuolization of the hepatocytes were increased in dosed groups of males and females. Based on these effects the LOAEL Value for the test chemical can be considered to be 30 mg/kg/day when the test chemical was dosed orally to mice for 3 months.

All of the above studies were supported by the liver toxicity of the test chemical was evaluated after a 5-day exposure. The test chemical was prepared for oral gavage administration in corn oil (Welch, Holme & Clark Co., Inc. Newark, NJ; Lot# 12-542) to deliver the chemical at doses of 0, 1, 6, 20, 60, or 120 mg/kg/day/chemical for five consecutive days to 5 male F344/N rats in a volume of 2.5-mL/kg body weight. Blood was collected the retroorbital plexus from animals anesthesized with CO2 and methemoglobin concentration determined by the method of Evelyn and Malloy (1938). Disodium hydrogen phosphate, potassium dihydrogen phosphate, acetic acid aqueous sodium cyanide and aqueous potassium ferricyanide were purchased from Sigma Chemical Company. Rats were euthanized by CO2 gas. The liver was harvested and weighed, and then a representative section from the midline of the left lateral hepatic lobe was collected and fixed in 10% neutral buffered formalin for histology. Hematoxylin and eosin (H&E)-stained sections (5 μm) were examined by a board certified pathologist. Three samples (approximately 3–4 mm cubes) were dissected from the remaining left lateral hepatic lobe, and flash frozen in liquid nitrogen and stored at −80°C until RNA extraction. RNA was extracted from frozen samples using the Invitrogen PureLink Mini kit (Invitrogen cat# 12183-018A, Carlsbad, CA) according to the manufacturer's protocol. RNA concentration and quality were measured on a Bioanalyzer (Agilent Technologies, Santa Clara, CA). Samples were aliquoted and stored at −80°C until they were analyzed for gene expression studies. All animals survived the 5-day exposure period. Methemoglobin levels were not increased in treated rats. At the end of the 5-day test chemical exposure, mean liver weight 6.2 ± 0.29, 6.3 ± 0.40, 6.2 ± 0.23, 6.2 ± 0.32, 6.7 ± 0.30, 7.9* ± 0.37 grams (*p<0.05), respectively. In the liver of test chemical treated rats, there were increased incidences of individual cell death in the 20, 60 and 120 mg/kg dose groups. Individual cell death was typically observed in the centrilobular to midzonal area, as shrunken, eosinophilic round bodies and cells with brightly eosinophilic cytoplasm and pyknotic, karyorrhectic nuclear debris. Eosinophilic globules were occasionally observed within the cytoplasm of adjacent hepatocytes. Taken together, these changes were suggestive of apoptosis. Grading was based on the number of dead cells observed within each lobule and the number of lobules involved; minimal lesions typically had 3 -5 dead cells in about half of the hepatic lobules; mild lesions typically had 5–8 cells with the involvement of most lobules. Two animals in the 60 mg/kg dose group and two animals in the 120 mg/kg dose group had an increase in the number of mitotic figures in the liver. Mitotic figures were common in the control animals due to their young age, and some 20X fields contained 1-3 mitotic figures. In comparison, animals that had minimally increased mitotic figures contained between 3 and 10 mitotic figures per 20X field.  All lesions observed were considered to be of minimal to mild severity. Hence, based on these observations the LOAEL can be considered to be 20 mg/kg/day.

Based on the available results, Administration of the test chemical resulted in increased incidences of nonneoplastic lesions of the liver and nasal cavity in male and female rats and mice; the kidney in male and female rats; the spleen and bone marrow in male and female rats and female mice; the lung in male and female mice; the forestomach in male rats and female mice; the mesenteric lymph node in male rats and female mice; and the olfactory lobe in male and female mice. Dimethyl-4-toluidine also caused hematologic toxicity and increases in methemoglobin levels in male and female rats and mice (as measured at 3 months).in males and females. The NOAEL value can be considered to be 6 mg/kg/day. Based on these effects, the test chemical is proposed to be classified under the category “STOT-RE2” as per CLP Regulation.

Repeated dose inhalation

Various studies have been reviewed to evaluate the toxicity potential of the test chemical when exposed repeatedly via inhalation route. These include in vivo experimental studies performed on rats for the test chemical. The results are mentioned below:

A study was performed according to OECD 412 to evaluate the toxicity potential of the test chemical when exposed to rats via inhalation route. Sprague Dawley rats were exposed to 5.6, 32.8, 67.6 ppm of the test chemical 6 hr/day, 5 days/week for 2 weeks with post exposure period of 2 weeks, control and high exposure groups. There were no deaths, and no changes in body weights, food consumption, clinical observations, or clinical chemistry. Methemoglobinemia was significantly increased across all exposure groups at both terminal and recovery necropsies in both sexes. Enlarged spleens, increased production of red cells in the spleen, bone marrow, and liver, and other hematology changes were consistent with induction of hemolytic anemia. Kidney effects were considered secondary to hemolytic anemia. This condition was not completely reversed at the end of the 14 day recovery period. A "no effect level" was not established under the conditions of this study. However, the lowest exposure level of 5.6 ppm was considered a "no adverse effect level" because the increase in methemoglobin was not accompanied by adverse histopathology or clinical signs.

 

This result is supported by a study whose objective and focus of the toxicity was to evaluate the toxicity by a 4-wk inhalation exposure to the test chemical. The study was performed according to OECD 412 Guidelines.Groups of 10 rats/sex were nose-only exposed to mean analytical concentrations of 19.1, 115.1, and 702.3 mg/m3 using an exposure regimen of 6 h/day and 20–22 exposures within a time period of 4 wk. These concentrations were selected based on results from a repeated 5×6 h/day pilot study using concentrations of 27.1, 104.8, 381.6, and 1283.7 mg/m3. Animals subjected to necropsy were sacrificed 1 day after the last exposure. During the study, animals were observed before and after each exposure for signs of toxicity. Body weights were recorded twice/week. Rectal temperatures were measured shortly after exposure using a rectal probe repetitively during the course of study.Ophthalmic exams were performed on 5 animals/sex/group before initiation of dosing and prior to termination. Blood was collected retro-orbitally at study midterm in 5 rats/sex/group for selected hematology and clinical pathology endpoints. The following serum clinical chemistry parameters were evaluated in all rats: alanine aminotransferase (ALAT), albumin, alkaline phosphatase, aspartate aminotransferase (ASAT), blood urea nitrogen, cholesterol, creatine kinase, creatinine, glucose, glutamate dehydrogenase (GLDH), lactate dehydrogenase (LDH), leucine arylamidase (LAP), serum protein electrophoresis, total bilirubin, total protein, triglycerides, and the electrolytes calcium, inorganic phosphorus, magnesium, sodium, potassium, and chloride. In hepatic tissue, cytochrome P-450, N-demethylase, O-demethylase, and triglycerides were determined. All animals of the 4-wk study were sacrificed by exsanguination (heart puncture) under diethyl ether anesthesia. Organ weights were recorded for adrenals, brain, heart, kidneys, liver, lungs, ovaries, spleen, testes, thymus, and thyroid. Organs and tissues were preserved in 10% neutral-buffered formalin. Prior to trimming, the head was decalcified in formic acid.The organs/tissues were embedded in paraffin, cut (approximately 5μm), and stained with hematoxylin and eosin (H&E). The nasal cavity was transversely sectioned at four levels largely consistent with the procedure described byYoung (1981) and the larynx was sectioned at the pouch level according to Lewis (1981). The following hematology parameters were evaluated in all rats at terminal sacrifice: red blood cell count, red blood cell morphology, hematocrit, hemoglobin, mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), mean cell volume (MCV), leukocyte count, reticulocyte counts, Heinz bodies, platelet (thrombocyte) count, prothrombin time. Exposure up to 115.1 mg/m3 was tolerated without clinical signs and mortality. Rats exposed to 702.3 mg/m3 displayed signs, most of them related to a cyanotic appearance, respiratory distress and breathing sounds, bradypnea, nasal discharge and irritation, and reduced motility.Rectal temperatures were significantly decreased at 115.1 mg/m3 and above. Dark and enlarged spleens occurred at 702.3 mg/m3. At this concentration, prominent treatment-related effects included methemoglobinemia, reticulocytosis, red blood cells with Heinz bodies, decreased hemoglobin, hematocrit, and red blood cell counts. Borderline evidence of erythrocytotoxicIty was noticed at 115.1 mg/m3 (based on a minimal increase in Heinz bodies). Spleen and liver weights were significantly increased at 702.3 mg/m3, whereas the thymus weight was decreased at 115.1 mg/m3 and above. Microscopic changes were found in the spleen (hemosiderosis) at 702.3mg/m3.Anatrophy of the olfactory epithelium in the nasal cavities occurred at 115.1mg/m3 and above. Clinical pathology revealed changes pathognostic of hepatic effects, although microscopic examinations did not reveal any specific changes. The no-observed-adverse-effect level (NOAEL) of the 4-wk study was 19.1 mg/m3 and is based on the predominant atrophic changes of the olfactory epithelium and the minimal to borderline erythrocytotoxic effects at 115.1 mg/m3.

Based on the available results and applying the weight of evidence approach, the NOAEC can be considered to be 19.1 mg/m3

Repeated Dose Dermal

A short term dermal toxicity study need not be conducted because exposure of humans via dermal in production and/or in use is highly unlikely based on the thorough and rigourous risk assessment provided. The acute dermal LD50 of the test chemical (as per section 7.2.3) was greater than 2000 mg/kg. Also the experimental in vivo skin irritation study suggests that the test chemical was not irritating to human skin. Hence this endpoint can be considered for waiver.

Justification for classification or non-classification

Based on the available results, Administration of the test chemical resulted in increased incidences of nonneoplastic lesions of the liver and nasal cavity in male and female rats and mice; the kidney in male and female rats; the spleen and bone marrow in male and female rats and female mice; the lung in male and female mice; the forestomach in male rats and female mice; the mesenteric lymph node in male rats and female mice; and the olfactory lobe in male and female mice. The test chemical also caused hematologic toxicity and increases in methemoglobin levels in male and female rats and mice (as measured at 3 months).in males and females. The NOAEL value can be considered to be 6 mg/kg/day. Based on these effects, the test chemical is proposed to be classified under the category “STOT-RE2” as per CLP Regulation.