N,N-diethylaniline

Endpoint summary

Description of key information

Repeated dose toxicity: Oral

In a subacute repeated-dose toxicity study, Wistar rats (male and female; number unspecified) were administered the test chemical via gavage at 0, 10, 50 or 250 mg/kg-bw/day, 7 days/week for 28 days. No mortalities were observed. No changes in body weight, food, and water consumption were reported. Clinical signs of toxicity consisted of increased frequency of respiratory sounds in males at 50 mg/kg-bw/day, and increased frequency of respiratory sounds and salivation in females at 250 mg/kg-bw/day. Hematological effects (decreased red cell counts, decreased hemoglobin concentrations, decreased packed cell volume (PCV) in both sexes and increased mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) in females) were reported at all doses (dose-response not specified). Histological effects were reported for the liver and spleen. In the liver, hemosiderosis of the Kupffer cells at 10 mg/kg-bw/day and extra medullary hematopoeisis at 50 and 250 mg/kg-bw/day were observed. In the spleen, hemosiderosis, extramedullary hematopoiesis and splenic hyperemia were reported at 10 mg/kg-bw/day. Swollen spleens were observed at 50 and 250 mg/kg-bw/day. Increased absolute and relative weights and black pigmentation of the spleen were also reported at 10mg/kg-bw/day. At 50 and 250 mg/kg-bw/day, hyperbilirubinemia, polychromasia were reported, and at 250 mg/kg-bw/day, decreased potassium levels, histopathological findings in the kidneys of both sexes, black pigmentation in the kidneys of females, and increased albumin levels in males were reported. Dose-response and statistical significance were not indicated for any of these observed effects. Based on hematological and histopathological changes in the spleen and liver consistent with hemolytic anemia observed the Low observed adverse effect level (LOAEL) for the test chemical using Wistar rats was considered to be 10 mg/kg-bw/day.

Repeated dose toxicity: Inhalation

The test chemical is likely to have a no observed adverse effect concenration (NOAEC) of 9.2 mg/m3 air upon repeated exposure by inhalation route to rats.

Repeated dose toxicity: Dermal

The acute dermal toxicity value for N,N-diethylaniline (CAS no 91-66-7)  (as provided in section 7.2.3) is >2000 mg/kg body weight. Considering this, the end point for repeated dermal toxicity is considered as waiver.

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:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from secondary literature

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Subacute repeated dose oral toxicity study was performed to determine the toxic nature of the test chemical.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 10, 50 or 250 mg/kg-bw/day
Basis:
no data

No. of animals per sex per dose:

unspecified

Control animals:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs of toxicity consisted of increased frequency of respiratory sounds in males at 50 mg/kg-bw/day, and increased frequency of respiratory sounds and salivation in females at 250 mg/kg-bw/day.

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortalities observed.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematological effects (decreased red cell counts, decreased hemoglobin concentrations, decreased packed cell volume (PCV) in both sexes and increased mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) in females) were reported at all dose

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histological effects were reported for the liver and spleen.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

HISTOPATHOLOGY:
Histological effects were reported for the liver and spleen. In the liver, hemosiderosis of the Kupffer cells at 10 mg/kg-bw/day and extra medullary hematopoeisis at 50 and 250 mg/kg-bw/day were observed. In the spleen, hemosiderosis, extramedullary hematopoiesis and splenic hyperemia were reported at 10 mg/kg-bw/day. Swollen spleens were observed at 50 and 250 mg/kg-bw/day. Increased absolute and relative weights and black pigmentation of the spleen were also reported at 10mg/kg-bw/day. At 50 and 250 mg/kg-bw/day, hyperbilirubinemia, polychromasia were reported, and at 250 mg/kg-bw/day, decreased potassium levels, histopathological findings in the kidneys of both sexes, black pigmentation in the kidneys of females, and increased albumin levels in males were reported.

Dose descriptor:

LOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on hematological and histopathological changes in the spleen and liver consistent with hemolytic anemia

Critical effects observed:

not specified

Conclusions:

The Low observed adverse effect level (LOAEL) for the test chemical using Wistar rats was considered to be 10 mg/kg-bw/day

Executive summary:

In a subacute repeated-dose toxicity study, Wistar rats (male and female; number unspecified) were administered the test chemical via gavage at 0, 10, 50 or 250 mg/kg-bw/day, 7 days/week for 28 days. No mortalities were observed. No changes in body weight, food, and water consumption were reported. Clinical signs of toxicity consisted of increased frequency of respiratory sounds in males at 50 mg/kg-bw/day, and increased frequency of respiratory sounds and salivation in females at 250 mg/kg-bw/day. Hematological effects (decreased red cell counts, decreased hemoglobin concentrations, decreased packed cell volume (PCV) in both sexes and increased mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) in females) were reported at all doses (dose-response not specified). Histological effects were reported for the liver and spleen. In the liver, hemosiderosis of the Kupffer cells at 10 mg/kg-bw/day and extra medullary hematopoeisis at 50 and 250 mg/kg-bw/day were observed. In the spleen, hemosiderosis, extramedullary hematopoiesis and splenic hyperemia were reported at 10 mg/kg-bw/day. Swollen spleens were observed at 50 and 250 mg/kg-bw/day. Increased absolute and relative weights and black pigmentation of the spleen were also reported at 10mg/kg-bw/day. At 50 and 250 mg/kg-bw/day, hyperbilirubinemia, polychromasia were reported, and at 250 mg/kg-bw/day, decreased potassium levels, histopathological findings in the kidneys of both sexes, black pigmentation in the kidneys of females, and increased albumin levels in males were reported. Dose-response and statistical significance were not indicated for any of these observed effects. Based on hematological and histopathological changes in the spleen and liver consistent with hemolytic anemia observed the Low observed adverse effect level (LOAEL) for the test chemical using Wistar rats was considered to be 10 mg/kg-bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Data is from secondary source

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:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Principles of method if other than guideline:

WoE derived based on the experimental data from various test chemicals

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: 1. Wistar; 2. Sprague Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

1. TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Specific-pathogen-free.
- Age at study initiation: approximately 2-3 months old
- Weight at study initiation: average body weight was 240 g
- Fasting period before study: 5 days
- Housing: polycarbonate cages containing bedding material (low-dust wood shavings)
- Diet (e.g. ad libitum): KLIBA 3883 pellets maintenance diet; PROVIMI KLIBA SA; ad libitum
- Water (e.g. ad libitum): municipality tap water in drinking bottles; ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ≈22°C
- Humidity (%): 40%–60%
- Air changes (per hr): Not available.
- Photoperiod (hrs dark / hrs light): 12-h light/dark cycle

2. No data

Route of administration:

inhalation: gas

Type of inhalation exposure:

other: 1. nose only; 2. Whole body

Vehicle:

not specified

Details on inhalation exposure:

1. GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:No data
- Method of holding animals in test chamber:No data
- Source and rate of air:No data
- Method of conditioning air: No data
- System of generating particulates/aerosols: digitally controlledmassflowcontrollers
- Temperature, humidity, pressure in air chamber:pproximately 23°C & Humidity 55%
- Air flow rate: The total flow rate directed into the nose-only exposure chamber was 30 l/min.
- Method of particle size determination: liquid aniline measures of bubbler: diameter: ≈2 cm in the 10, 30, and 90 mg/m3 groups and 4.5 cm in the 270 mg/m3, height of liquid level: ≈5 cm

TEST ATMOSPHERE
- Brief description of analytical method used:bubbler containing liquid aniline measures of bubbler: diameter: ≈2 cm in the 10, 30, and 90 mg/m3 groups and 4.5 cm in the 270 mg/m3, height of liquid level: ≈5 cm)These bubblers were maintained at 25°C with a digitally controlled thermostat Air flows passed through the liquid aniline were controlled by a calibrated gas-metering device (digitally controlled mass flow controllers) and ranged from 0.12 l/min to 3.4 l/min in the low and high group, respectively. This atmosphere was subsequently diluted by conditioned, dry air. The total flow rate directed into the nose-only exposure chamber was 30 l/min. Air flows passed through the liquid aniline were controlled digitally by calibrated mass flow controllers
Rats of the control group were exposed nose-only to dry, filtered air only.
The inhalation chamber had a volume of 7.6 l and was suitable to accommodate 40 rats. The air flow rate supplied into and extracted from the chamber provided a slight positive balance of air flow toward the rats’ breathing zone, and was maintained at 0.75 l/min/exposure port.

2. No data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

1. Exposure atmospheres were characterized by using a gas chromatographic technique (Hewlett-Packard GC 5890 equipped with a flame ionization
detector, split/splitless injector capillary column HPUltra 50+, length: 30 m, ID: 0.32 mm, film thickness 0.17 mm, autosampler HP 7673, and an HP 3365 Work-Station for data processing). For GC analyses, samples were taken from the vicinity of the rats’ breathing zone three times per exposure day using two impingers (in-line) filled with toluene. Between the glass bubblers and the gas-metering device (digital flow controller) a cool-trap was used to scrub volatile constituents from the sampled air. For calibrations, the test substance served as reference material. The sampling flow rate was 0.5 l/min. The sample volumes were 50, 20, 10, and 10 l/sample in the 10, 30, 90, and 270 mg/m3 group, respectively. In the last two groups, the temporal stability of concentrations of aniline in the chambers was monitored continuously using a Compur Total Hydrocarbon Analyzer (Compur, Munich, Germany). Chamber temperature and humidity were measured electronically, and mean values were approximately 23°C and 55%, respectively.


2. No data

Duration of treatment / exposure:

1. 28 days
6h/day head-only to aniline vapor

2. 14 days

Frequency of treatment:

1. 5 days/week for 14 days followed by a post-exposure period up to 28 days
2. 6 hours/day, 5 days/week for 14 days

Remarks:

Doses / Concentrations:
10, 30, 90, and 270 mg/m3 / 1
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
9.2, 32.4, 96.5, and 274.9 mg/m3 / 1
Basis:
analytical conc.

Remarks:

0, 5.6, 32.8 or 67.6 ppm (0.03, 0.18, 0.37 mg/L, respectively) / 2

No. of animals per sex per dose:

1. 30 Male
2. No data

Control animals:

yes

Details on study design:

1. Although female rats are often shown to be more susceptible to the formation of MetHb and the development of anemia than male rats, this study utilized male rats only to allow a better comparison with existing mechanistic studies.

Male Wistar rats were exposed nose only to aniline vapor in targeted concentrations of 10, 30, 90, and270 mg/m3 for 6h/day, 5days/week for 2 weeks (days 0–11), followed by a 2-week post-exposure period (up to day 28).
The control group was exposed to conditioned, dry air under otherwise identical conditions.
Serial sacrifices for specialized examinations were performed on days 0, 4, 11, 14, and 28 to address the time-course and reversibility of changes.
The selection of specific endpoints considered publications of previous studies with aniline and its structural analogs.

2. - Dose selection rationale:Not available
- Rationale for animal assignment (if not random):Not available
- Rationale for selecting satellite groups:Not available
- Post-exposure recovery period in satellite groups:Not available
- Section schedule rationale (if not random):Not available

Observations and examinations performed and frequency:

1. DETAILED CLINICAL OBSERVATIONS: Yes
Data generated on the clinical chemistry focused on potential hepatotoxicity and erythrocyte (hemoglobin) catabolism by the end of the exposure and post-exposure periods. Statistical comparisons did not reveal any consistent concentration–response relationship considered to be of pathodiagnostic relevance except slightly altered bilirubin serum concentrations at 90 and 270 mg/m3. Bilirubin concentration was increased dose dependent on day 14 (up to 1.4 fold over control) and dose dependently decreased by the end of the post-exposure period (up to 40%).
Some electrolytes (calcium, magnesium) were statistically significantly decreased at 30 mg/m3 and above, especially at the end of the post-exposure period. Because of the lack of a clear dose-dependence from 30 to 270 mg/m3 - in comparison to the control and 10 mg/m3 groups - these changes are considered without biological significance. They are considered to be secondary to changes in the concentrations of negatively charged
counterions (anion gap), such as plasma proteins or bicarbonate, rather than reflecting any specific, aniline-induced disturbance in electrolyte homeostasis.
A concentration-dependent and time-dependent increase in the total splenic iron content were observed in rats exposed to 90 and 270 mg/m3, when compared to the controls. Rhe increase in total iron and splenomegaly are related. In contrast, the maximum iron content in liver tissue homogenates was transient, and a slight increase was observed at 270 mg/m3 only. Based on a gram tissue level comparison, the maximum accumulation of iron in the liver and spleen exceeded the control levels by approx. 60% and approx. 500%, respectively.
Lipid peroxidation, measured as the sum of malondialdehyde and 4-hydroxy-2(E)-nonenal, was significantly increased at 90 and 270 mg/m3 three to four-fold over the control group. Despite the marked increase in lipid peroxidationfrom exposure day 0 to exposure day 4, a time-dependent progression between the later exposure days was not apparent. Changes subsided toward the level of the control group at the end of the post-exposure period, i.e., the aniline exposure groups were indistinguishable from the control. During the exposure period, the increase of splenic lipid peroxidation and total iron was highly correlated (r2 = 0.93), whereas this high degree of correlation ceased to exist at the end of the post-exposure period.
The determination of ferritin and glutathione peroxidase (GPx) in the tissue homogenates of the spleen did not attain statistical significance, when compared with the respective controls. However, despite the lack of any consistent concentration-dependence or time-dependence, GPx activity showed a tendency toward increase at the high exposure level.

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly; Mondays and
Fridays and once weekly during the post-exposure period

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: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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: No data

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

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 4,11 & 14.
- Anaesthetic used for blood collection: No data
- Animals fasted: no data
- How many animals:
- Parameters checked in table [No.?] were examined.
Hematological parameters were significantly affected at 90 and 270 mg/m3 starting on day 4, with maximum effects on days 11 and 14. They were characterized by a decrease up to 16% in hemoglobin (Hb), red blood cell count (RBC, up to 24%), and hematocrit (HCT, up to 20%), and an increase in reticulocyte counts (RC, up to 6.3fold over control) and erythrocytes containing Heinz bodies (day 4: high dose. 513/1000 cells vs 0/1000 in the control group; day 14: high dose. 266/1000 cells vs 0/1000 in the control group).
The RC observed in controls on day 0 (56/1000) were twice as high as those observed at the subsequent time points. This change might be attributable to the immobilization stress caused by restraint of non-acclimatized rats to exposure tubes and the associated increased discharge of mature reticulocytes from the bone marrow.
On day 11 only rats exposed to 270 mg/m3 showed a significantly increased mean corpuscular hemoglobin (MCH, 1.1 fold over control) and mean corpuscular volume (MCV, 1.2 fold). Furthermore, on day 11 the red blood cells in some rats exhibited hypochromia and anisocytosis.
With regard to the time-course, the hematological changes were maximal on the last exposure day (day 11) and showed some decrease in magnitude on the third post-exposure day (day 14); then, with the exception of the RBC, MCH, and MCHC, hematological changes subsided to the levels of the control group after the 2 week post-exposure.
Thrombocytes were mildly, although significantly increased at the end of the 2-week exposure period (data not shown). Conclusive changes in the total leukocyte counts and leukocyte differentials did not occur at any time point (data not shown).
In the blood sampled and determined immediately after cessation of exposure, MetHb was concentration-dependent, and then was significantly increased at 90 mg/m3 (days 4 and 11, up to approx. 2.5% vs. approx 1% in the control group) and at 270 mg/m3 (days 0, 4, 11, up to approx. 14% vs. approx. 1% in the control group). The MetHb concentrations from blood samples taken in the post-exposure period were indistinguishable from the control.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: No data
- How many animals:
- Parameters checked in table [No.?] were examined.
Data generated on the clinical chemistry focused on potential hepatotoxicity and erythrocyte (hemoglobin) catabolism by the end of the exposure and post-exposure periods. Statistical comparisons did not reveal any consistent concentration–response relationship considered to be of pathodiagnostic relevance except slightly altered bilirubin serum concentrations at 90 and 270 mg/m3. Bilirubin concentration was increased dose dependent on day 14 (up to 1.4 fold over control) and dose dependently decreased by the end of the post-exposure period (up to 40%).
Some electrolytes (calcium, magnesium) were statistically significantly decreased at 30 mg/m3 and above, especially at the end of the post-exposure period. Because of the lack of a clear dose-dependence from 30 to 270 mg/m3 - in comparison to the control and 10 mg/m3 groups - these changes are considered without biological significance. They are considered to be secondary to changes in the concentrations of negatively charged
counterions (anion gap), such as plasma proteins or bicarbonate, rather than reflecting any specific, aniline-induced disturbance in electrolyte homeostasis.
A concentration-dependent and time-dependent increase in the total splenic iron content were observed in rats exposed to 90 and 270 mg/m3, when compared to the controls. Rhe increase in total iron and splenomegaly are related. In contrast, the maximum iron content in liver tissue homogenates was transient, and a slight increase was observed at 270 mg/m3 only. Based on a gram tissue level comparison, the maximum accumulation of iron in the liver and spleen exceeded the control levels by approx. 60% and approx. 500%, respectively.
Lipid peroxidation, measured as the sum of malondialdehyde and 4-hydroxy-2(E)-nonenal, was significantly increased at 90 and
270 mg/m3 three to four-fold over the control group. Despite the marked increase in lipid peroxidationfrom exposure day 0 to exposure day 4, a time-dependent progression between the later exposure days was not apparent. Changes subsided toward the level of the control group at the end of the post-exposure period, i.e., the aniline exposure groups were indistinguishable from the control. During the exposure period, the increase of splenic lipid peroxidation and total iron was highly correlated (r2 = 0.93), whereas this high degree of correlation ceased to exist at the end of the post-exposure period.
The determination of ferritin and glutathione peroxidase (GPx) in the tissue homogenates of the spleen did not attain statistical significance, when compared with the respective controls. However, despite the lack of any consistent concentration-dependence or time-dependence, GPx activity showed a tendency toward increase at the high exposure level

2. CAGE SIDE OBSERVATIONS: Yes
BODY WEIGHT: Yes
FOOD CONSUMPTION:Yes
HAEMATOLOGY: Yes
CLINICAL CHEMISTRY: Yes

Sacrifice and pathology:

1. GROSS PATHOLOGY: Yes
All animals were euthanized by complete exsanguination (heart puncture) after intraperitoneal sodium-pentobarbital injection (Narcoren, Merial GmbH, Hallbergmoos, Germany). All rats were given a gross-pathological examination.
Weights were recorded for brain, liver, lung, spleen, and testes at interim sacrifices (days 0, 4, and 11) and, in addition, heart, kidneys, and thymus at post-exposure sacrifices (days 14/15 and 28). For the heart, kidneys, and thymus no appreciable time-related effects were expected to occur. Therefore, the weights of these organs were determined only at the time points where the maximum effect, including reversibility, can be evaluated

HISTOPATHOLOGY: Yes
From the additional 5 rats/group that were sacrificed on day 15, selected organs and tissues (spleen, femur, sternum, liver, lung) were preserved in 10% neutral-buffered formalin or Davidson’s solution (testes) for histopathology. The lung was intratracheally instilled with the fixative under 20 cm H2O pressure. Osseous tissues were first decalcified and then, as for all other organs, embedded in Paraplast. All slides were stained with hematoxylin and eosin (H&E).
For a better appreciation of the degree of hemosiderosis, a specific iron stain (Prussian blue stain according to Perls) was prepared for formol-fixed sections of the liver and spleen.

2. GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

1. All animals survived the duration of study. Concentrations up to and including 30 mg/m3 were tolerated without clinical signs.
Rats exposed to 90 mg/m3 and above were cyanotic (blue discoloration of the skin in areas easily observed), whereas those exposed to 270 mg/m3 also displayed tachypnea, labored breathing patterns, increased salivation, and an ungroomed hair-coat. All signs disappeared toward the following day and did not show any exacerbation during the course of the study. Rectal temperatures measured shortly after cessation of exposure on days 0, 4, and 11 were at all-time points indistinguishable among the groups (range of means in the control group: 37.3°–38.6°C, aniline exposure groups: 36.6°–38.4°C).
Body weights were not statistically significantly affected in any group during the course of the study.
The weights of liver (Table 3), brain, lung, testes, heart, kidneys, and thymus did not show treatment-related effects (data not shown). Absolute and relative spleen weights were significantly increased in a time and dose dependent matter. In the group treated with 270 mg aniline/m3 maximum increase of 2.2 fold over the spleen weights of the control (day 11) was observed.
Gross necropsy findings of rats exposed to 90 mg/m3 and above provided evidence of dark discolorations and enlargement of spleens at all sacrifices beyond the first exposure week.
Some discoloration of lungs was found; however, no consistent time dependence or concentration-dependence existed. An increased incidence of discoloration of the thymus, including involution, and of the testes occurred at 270 mg/m3 (days 11 and 14).

Statistics:

1. Body weights, hematology, and clinical pathology data were compared using either
(1) the Kruskal-Wallis test and the adjusted U-test or adjusted Welsh-test as the post hoc test or
(2) One-way analysis of variance (ANOVA) and the Dunnett test as the post hoc tests for nonparametric and parametric analyses, respectively (SAS 6.12-routines).
Specialized endpoints (GPx, ferritin, iron) were analyzed with one-way ANOVA and the Tukey-Kramer post hoc test (BCTIC).
Histopathological findings were compared with the concurrent control using Fisher’s exact test.
The proportion of erythrocytes with Heinz bodies was transformed prior to ANOVA analysis using the arcsine square root function. This is appropriate for percentages and proportions, because the transformed data more closely approximate a normal distribution than the non-transformed proportions.
Transformed data were analyzed separately for normality of distribution. For all tests, the criterion for statistical significance was set at p < 0.05.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

1. No clinical signs

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1. No significant change in body weight

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):

1. Subsequent increases in erythrocyte damage and turnover, including anemia, RBC morphological alterations.

Clinical biochemistry findings:

effects observed, treatment-related

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:

effects observed, treatment-related

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

2. Haematology-significant increases in methemoglobinemia at both terminal and recovery necropsies in both sexes across all concentrations.Enlarged spleens, increased red blood cells in the spleen, bone marrow and liver, and other hematological changes were consistent with the induction of hemolytic anemia at concentrations > 5.6 ppm (0.03 mg/L).Observed kidney effects were considered secondary to hemolytic anemia.

Dose descriptor:

NOAEC

Remarks:

1

Effect level:

9.2 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: slight increase in extra medullary hematopoiesis; this concentration is considered NOAEC for erythro toxicity, iron accumulation and lipid peroxidation

Dose descriptor:

LOAEC

Remarks:

1

Effect level:

32.6 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: slight increase in extra medullary hematopoiesis

Dose descriptor:

NOAEC

Remarks:

2

Effect level:

0.03 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No deaths or changes in body weight, food consumption, clinical observations or clinical chemistry parameters were reported for any exposure level

Dose descriptor:

LOAEC

Remarks:

2

Effect level:

0.18 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on hematological changes consistent with hemolytic anemia

Critical effects observed:

not specified

Conclusions:

The test chemical is likely to have a no observed adverse effect concenration (NOAEC) of 9.2 mg/m3 air upon repeated exposure by inhalation route to rats.

Executive summary:

Data available for the varios test chemicals was reviewed to determine the toxic nature of the test chemical upon repeated exposure by inhalation route. The studies are as mentioned below:

A subacute inhalation toxicity study of the test chemical was conducted inmale Wistar rats for 28 days. Clinical signs of toxicity, body weights, hematology, and clinical chemistry tests, including total iron in liver and spleen, splenic lipid peroxidation, organ weights, gross and histological changes in target organs were recorded. No mortality was observed during the study. The changes observed included anemia, red blood cell morphological alterations (e.g., Heinz bodies), decreased hemoglobin and hematocrit, reticulocytosis, and effects on the spleen (splenomegaly, hemosiderin accumulation, and increased hematopoietic cell proliferation). The total content of iron in spleen homogenates increased in a concentration-dependent and time-dependent manner with increasing duration of exposure. The maximum accumulation of iron in the liver and spleen exceeded the respective control levels by ≈60% and ≈500%, respectively. The Lowest observed adverse effect cocentration (LOAEC) was observed in rat at 32.6 mg/m³ air (analytical) and the No observed adverse effect concentration (NOAEC) wasconsidered to be at 9.2 mg/m³ air (analytical) respectively.

In a repeated-dose inhalation toxicity study, male and female Sprague-Dawley rats were exposed to the test chemical via inhalation route of exposure at 0, 5.6, 32.8 or 67.6 ppm (0.03, 0.18, 0.37 mg/L, respectively). During the study, the treated animals were observed for clinical signs, mortality, body weight changes, changes in food consumption, hematology and clinical chemistry observations and were subjected to gross pathology and histopatholgy. Exposure to 5.6, 32.8, or 67.6 ppm test material vapor under the conditions of this study induced hemolytic anemia without recovery in male and female rats. A no observed adverse effect concentation (NOAEL) of 5.6 ppm (0.03 mg/l) and low observed adverse effect concentration (LOAEL) of 32.8 ppm (0.18 mg/l) was determined based on a reported methemoglobin level.

Based on the observations, the test chemical is likely to have a no observed adverse effect concenration (NOAEC) of 9.2 mg/m³ air upon repeated exposure by inhalation route to rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

9.2 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Data is from authoritative source

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal, other

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Quality of whole database:

Waiver

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data available for the target chemical and various test chemicals was reviewed to determine the toxic nature upon repeated exposure. The studies are as mentioned below:

Repeated dose toxicity: Oral

In a subacute repeated-dose toxicity study, Wistar rats (male and female; number unspecified) were administered the test chemical via gavage at 0, 10, 50 or 250 mg/kg-bw/day, 7 days/week for 28 days. No mortalities were observed. No changes in body weight, food, and water consumption were reported. Clinical signs of toxicity consisted of increased frequency of respiratory sounds in males at 50 mg/kg-bw/day, and increased frequency of respiratory sounds and salivation in females at 250 mg/kg-bw/day. Hematological effects (decreased red cell counts, decreased hemoglobin concentrations, decreased packed cell volume (PCV) in both sexes and increased mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) in females) were reported at all doses (dose-response not specified). Histological effects were reported for the liver and spleen. In the liver, hemosiderosis of the Kupffer cells at 10 mg/kg-bw/day and extra medullary hematopoeisis at 50 and 250 mg/kg-bw/day were observed. In the spleen, hemosiderosis, extramedullary hematopoiesis and splenic hyperemia were reported at 10 mg/kg-bw/day. Swollen spleens were observed at 50 and 250 mg/kg-bw/day. Increased absolute and relative weights and black pigmentation of the spleen were also reported at 10mg/kg-bw/day. At 50 and 250 mg/kg-bw/day, hyperbilirubinemia, polychromasia were reported, and at 250 mg/kg-bw/day, decreased potassium levels, histopathological findings in the kidneys of both sexes, black pigmentation in the kidneys of females, and increased albumin levels in males were reported. Dose-response and statistical significance were not indicated for any of these observed effects. Based on hematological and histopathological changes in the spleen and liver consistent with hemolytic anemia observed the Low observed adverse effect level (LOAEL) for the test chemical using Wistar rats was considered to be 10 mg/kg-bw/day.

This is further supported by the data avaiable from various test chemicals as-

Repeated dose oral study was conducted to evaluate the histopathologic changes in the  specific target organs following the oral administration of the test chemical. The test chemical was administered to male Fischer 344 rats daily for 5,10 or 20 days orally through gavage needle. The study was designed to provide a histopathologic evaluation of specific target organs following oral administration of the test substance for 5,10 or 20 days. Histopathologic evaluation of selected tissues revealed splenic congestion, increased hematopoiesls and hemosiderosis, and bone marrow hyperplasia in aniline- treated animals. The low observed adverse effect level (LOAEL) for the test chemical in rats is considered to be 110 mg/kg bw/day.

In another repeated-dose toxicity study, the test chemical was administered at a dose concsntration of 0, 31, 62, 125, 250 or 500 mg/kg/day to 10 male and 10 female B6C3F1 mice per dose via gavage 5 days/week for 90 days. No mortality was observed. Dose-related increases in splenomegaly, and extramedullary hematopoiesis and hemosiderosis of the spleen were observed in the mice when given 31.25 mg/kg/day of the test chemical. Splenomegaly was reported as minimal in 4/10 mice, and extramedullary hematopoiesis and hemosiderois were reported as mild in 1/10 mice. Based on observed changes in the bone marrow and the spleen, the low observed adverse effect level (LOAEL) was considered to be 31 mg/kg/day in B6C3F1 mice.

In yet another repeated-dose toxicity study, the test chemical was administered at dose level of 0, 31.25, 62.5, 125, 250 or 500 mg/kg/day to 10 male and 10 female Fischer 344 rats per dose via gavage 5 days/week for 90 days. No mortality was observed. Splenomegaly was observed in all treated groups of rats. Hyperplasia of the bone marrow and hematopoiesis in the spleen were observed in all rats in a dose-related manner. In addition, decreased body weight gain was observed in male rats at 250 and 500 mg/kg/day. Based on observed changes in body weight, the bone marrow and the spleen, the low observed adverse effect level (LOAEL) was considered to be 31.25 mg/kg/day in Fischer 344 rats.

Repeated dose toxicity: Inhalation

A subacute inhalation toxicity study of the test chemical was conducted inmale Wistar rats for 28 days. Clinical signs of toxicity, body weights, hematology, and clinical chemistry tests, including total iron in liver and spleen, splenic lipid peroxidation, organ weights, gross and histological changes in target organs were recorded. No mortality was observed during the study. The changes observed included anemia, red blood cell morphological alterations (e.g., Heinz bodies), decreased hemoglobin and hematocrit, reticulocytosis, and effects on the spleen (splenomegaly, hemosiderin accumulation, and increased hematopoietic cell proliferation). The total content of iron in spleen homogenates increased in a concentration-dependent and time-dependent manner with increasing duration of exposure. The maximum accumulation of iron in the liver and spleen exceeded the respective control levels by ≈60% and ≈500%, respectively. The Lowest observed adverse effect cocentration (LOAEC) was observed in rat at 32.6 mg/m³ air (analytical) and the No observed adverse effect concentration (NOAEC) wasconsidered to be at 9.2 mg/m³ air (analytical) respectively.

In another repeated-dose inhalation toxicity study, male and female Sprague-Dawley rats were exposed to the test chemical via inhalation route of exposure at 0, 5.6, 32.8 or 67.6 ppm (0.03, 0.18, 0.37 mg/L, respectively). During the study, the treated animals were observed for clinical signs, mortality, body weight changes, changes in food consumption, hematology and clinical chemistry observations and were subjected to gross pathology and histopatholgy. Exposure to 5.6, 32.8, or 67.6 ppm test material vapor under the conditions of this study induced hemolytic anemia without recovery in male and female rats. A no observed adverse effect concentation (NOAEL) of 5.6 ppm (0.03 mg/l) and low observed adverse effect concentration (LOAEL) of 32.8 ppm (0.18 mg/l) was determined based on a reported methemoglobin level.

Repeated dose toxicity: Dermal

The acute dermal toxicity value for N,N-diethylaniline (CAS no 91-66-7) (as provided in section 7.2.3) is >2000 mg/kg body weight. Considering this, the end point for repeated dermal toxicity is considered as waiver.

Considering the available data for the target chemical and various other test chemicals, N,N-diethylaniline (CAS no 91-66-7) is likely to classify as a toxicant

upon repeated exposure by oral and inhlation route and can be considered to be STOT RE 2 as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Considering the available data for the target chemical and various other test chemicals, N,N-diethylaniline (CAS no 91-66-7) is likely to classify as a toxicant

upon repeated exposure by oral and inhlation route and can be considered to be STOT RE 2 as per the criteria mentioned in CLP regulation.