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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Principles of method if other than guideline:
Temperature and relative humidity were measured three times during exposure until 8 June 2011 instead of (at least) hourly.
The relative humidity was out of its target range on several occasions.
The temperature in the exposure chamber was above its target on the last exposure day for groups 3 and 4.
Exposure duration on the last exposure day (28 June 2011) was extended with 22 minutes for all groups due to blood sample collection.
These deviations are considered not to have affected the validity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Appearance : Pale yellow to colourless liquid
Molecular formula : C7H9N
Molecular weight : 107.15
Specific gravity : 0.986 g/cm3 at 20 ºC
Solubility in water : slight
Vapour pressure : 0.3 mm HG at 20 ºC
Melting point : -53 ºC
Boiling point : 195 ºC
CAS Reg. Number : 100-61-8
Purity : >93%
Batch number : Lot nr. 20110120
Total quantity : 500 g
Storage conditions : ambient temperature
Expiry date : 27 April 2012

Test animals

Species:
rat
Strain:
other: Crl:WI[WU]
Sex:
male/female
Details on test animals and environmental conditions:
After a 7-day acclimatization period to the laboratory conditions, the animals were allocated to the various exposure groups by computer randomisation proportionally to body weight.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Not applicable.
Details on inhalation exposure:
The animals were exposed to the test atmosphere in nose-only exposure units consisting of a 60-litre cylindrical column, surrounded by a transparent cylinder. Each unit holds 20 ports for animal exposure. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. The rats of each group were placed in an alternating order for sex. Only the nose of the rats protruded into the interior of the column. By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder, dilution of test atmosphere by air leaking from the animals’ thorax to the nose was avoided. Empty ports were used for test atmosphere sampling and measurement of temperature and relative humidity. The remaining ports were closed.
The test atmosphere originated from a liquid flow of the test material controlled by a syringe pump evaporated in a mass flow controlled stream of humidified air (mid and high concentration groups). The lowest concentration was generated by extraction of a part of the high concentration test atmosphere using a venturi and subsequently dilution with humidified air. The exposure unit for the control animals was supplied with a stream of humidified air only.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Three times on each exposure day, test atmosphere samples of 9, 3 and 1 L (at 273.16 K and 1013 Pa) were taken from the low, mid and high concentration test atmospheres, respectively, and led through impingers filled with a known amount of 0.05 M sulfuric acid. The content of N-methylaniline was determined by HPLC analysis and confirmed using XAD-2 cartridges.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 days/week (i.e. 20 exposure days over a 30-day study period).
Doses / concentrations
Remarks:
Doses / Concentrations:
15, 45 and 135 mg/m³
Basis:
other: Target concentrations
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
Details on study design:
No further details.
Positive control:
Not applied.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes
Animals were observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. A group-wise observation was made halfway through each exposure day.On working days, all cages were checked again in the afternoon, especially for dead or moribund animals. In weekends and on public holidays only one check per day was carried out. All abnormalities, signs of ill health, reaction to treatment and mortality were recorded.

BODY WEIGHT: Yes
Recorded one day before the start of exposure (day -4), prior to exposure on the first day (day 0) and twice weekly thereafter until day 29 before fasting and on day 30 after fasting for correct determination of relative organ weights.

FOOD CONSUMPTION: Yes
Measured per cage over three 7-day periods and one 8-day period, starting on the day of first exposure.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period blood samples were taken from the abdominal aorta.
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes, overnight
- How many animals: all surviving
- Parameters checked:
+ haemoglobin
+ methaemoglobin3
+ packed cell volume
+ red blood cell count
+ reticulocytes
+ total white blood cell count
+ differential white blood cell count
+ prothrombin time
+ thrombocyte count
+ mean corpuscular volume (MCV)
+ mean corpuscular haemoglobin (MCH)
+ mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- At the end of the treatment period blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation.
- Animals fasted: Yes, overnight
- How many animals: all surviving
- Parameters checked:
+ alkaline phosphatase activity (ALP) + bilirubin total + phospholipids
+ aspartate aminotransferase activity (ASAT) + cholesterol + calcium (Ca)
+ alanine aminotransferase activity (ALAT) + triglycerides + sodium (Na)
+ gamma glutamyl transferase activity (GGT) + inorganic phosphate + potassium (K)
+ total protein + fasting glucose + chloride (Cl)
+ albumin + creatinine
+ ratio albumin to globulin + urea
Sacrifice and pathology:
SACRIFICE: The day after the last exposure by exsanguination from the abdominal aorta under pentobarbital anaesthesia.
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
- adrenals
- bone marrow
- brain (including sections of cerebrum, cerebellum, and medulla/pons)
- heart
- lymph nodes from the hilar region of the lung
- oesophagus
- ovaries
- seminal vesicles
- spinal cord (cervical, mid-thoracic, and lumbar)
- stomach
- testes
- thymus
- thyroid
- uterus
- complete respiratory tract including nasopharyngeal tissues
- all gross lesions
Other examinations:
No further examinations.
Statistics:
Body weight data: ‘AnCova & Dunnett’s Test’ (abbreviation ANCDUN) with ‘Automatic’ as data transformation method.

Haematology, clinical chemistry and organ weight data: ‘Generalised Anova/Ancova Test’ (abbreviation GEN AN) with ‘Automatic’ as data transformation method.

Incidences of histopathological changes: Fisher’s exact probability test.

Tests are performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01.

Because numerous variables are subjected to statistical analysis, the overall false positive rate (Type I errors) is greater than suggested by a probability level of 0.05. Therefore, the final interpretation of results was based not only on statistical analysis but also on other considerations such as dose-response relationships and whether the results were significant in the light of other biological and pathological findings.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Occasionally effects were observed on breathing and incidentally cyanosis among female rats.
Mortality:
mortality observed, treatment-related
Description (incidence):
Occasionally effects were observed on breathing and incidentally cyanosis among female rats.
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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Except prothrombin time, all red blood cell parameters were affected by exposure to N-methylaniline.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Main finding: Bilirubin significantly increased in a concentration related manner for all exposed male groups and in females of the high concentration group.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute spleen weight was increased in all exposed female groups and in the mid and high concentration male groups. Relative spleen weight was increased in both male and female animals of the mid and high concentration groups
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related effects in the spleen and the bone marrow.
Histopathological findings: neoplastic:
not examined
Details on results:
Four groups of 5 male and 5 female animals were exposed to concentrations of 0 (control), 13.3 (± 0,75), 40,0 (± 2.8), or 115.9 (± 7.1) mg/m3. Exposure at these levels did not induce treatment-related changes in body weight gain or food consumption. Clinical abnormalities were not seen before the start of exposure . Occasionally, a decreased breathing frequency was seen at the group-wise observation during exposure, and two females of group 3 showed cyanosis just after exposure one day in the third week of the study.
Except prothrombin time, all red blood cell parameters were affected by exposure to N-methylaniline_EC 202-870-9. The concentration of red blood cells, haemoglobin, PCV, MCV and the percentage of reticulocytes were also significantly affected in animals of the low concentration group. The number of white blood cells, lymphocytes, basophils and monocytes were increased in female animals of the high concentration group.
Bilirubin in blood plasma was significantly increased dose-dependently and was significant for all exposed male groups and in females of the high concentration group. Creatinine in blood plasma was significantly decreased in female animals of the high concentration group.
Both relative and absolute spleen weights were increased in exposed male and female animals, which was significant for all mid and high concentration groups and the absolute spleen weight was also significantly increased in females of the low concentration group.
Macroscopic examination at necropsy did not show exposure-related gross pathology.
Microscopic examination showed treatment-related changes in the spleen and bone marrow. Changes in the bone marrow only occurred in males of
the high-concentration group, whereas changes in the spleen occurred in all exposed animals. The effects on the spleen, both at microscopical examination and on organ weight, and on the bone marrow at macroscopical examination are likely to be related to the effects on red blood cell parameters and on bilirubin concentrations in blood.

Effect levels

Dose descriptor:
LOAEC
Effect level:
13.3 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Comparison of endpoint values between the lowest concentration and the control group:

 

male

female

dose response

 

 

control

%SD

Lowest concentration

Stat signif.

control

%SD

Lowest concentration

Stat.signif.

 relation ?

body weights

47.18

10.72%

57.28

21%

-

20.34

3%

20.92

3%

-

No

food consumption

21.37

22.33

4%

-

15.7

16.03

2%

-

No

Haematology

RBC

9.774

3.1%

9.322

-5%

-

9.16

1.3%

8.636

-6%

+

Yes

Hb

10.28

2.2%

9.68

-6%

+

9.8

1.6%

9.34

-5%

+

No

MetHb

2.118

30.2%

0.835

-61%

+

1.89

15.8%

1.863

-1%

-

Yes

PCV

0.4818

2.8%

0.4572

-5%

+

0.4536

1.4%

0.436

-4%

+

No

MCV

49.3

0.5%

49.05

-1%

-

49.52

0.6%

50.49

2%

+

Yes

MCH

1.052

1.0%

1.039

-1%

-

1.07

2.0%

1.082

1%

-

Not consistent

MCHC

21.34

0.6%

21.18

-1%

-

21.61

1.7%

21.42

-1%

-

No

Reticulocytes

0.364

13.2%

0.62

70%

+

0.382

44.0%

1.008

164%

+

Not consistent

Thrombocytes

534.6

8.1%

575.4

8%

-

528

6.2%

556

5%

-

Not consistent

Prothrombine

53.18

45.4%

34.7

-35%

-

27.66

2.1%

28.02

1%

-

No

Bilirubine

1.72

2.3%

2

16%

+

2.24

25.0%

2.56

14%

-

Yes

Spleen (abs)

0.507

7.8%

0.5408

7%

-

0.3492

8.2%

0.3932

13%

+

Yes

Spleen (rel)

1.794

5.4%

1.821

2%

-

1.99

8.2%

2.181

10%

-

Yes

Spleen (hist)

very slight

slight

Not clear

Bone marrow (hist)

 

 

No

No

 

 

 

 

Applicant's summary and conclusion

Conclusions:
Results indicated that exposure at these levels did not induce treatment-related changes in body weight gain or food consumption, or clinical abnormalities except for incidental cyanosis just after exposure. The significant increase of both reticulocytes and bilirubin is related to the treatment related decrease of red blood cells (RBC) caused by exposure to N-methylaniline. However, the significant increase of reticulocytes also confirms that the bone marrow's ability to produce new blood cells remained intact. Further, the increased concentrations of bilirubin coincides with the histopathological observations in the spleen confirming an increased breakdown of red blood cells.
As treatment-related changes were seen in all groups exposed to N-methylaniline, a No-Observed-Adverse-Effect-Level (NOAEL) could not be determined. It should be noted that where changes in parameters or endpoints were significant, these were still relatively small. This indicates that the lowest actual concentration of 13.3 ± 0.75 mg/m³ is relatively close to the NOAEL for subacute exposure of rats to N-methylaniline by inhalation.
Executive summary:

The inhalation toxicity of N-methylaniline_EC 202-870-9 was studied in a sub-acute study with Wistar rats. Groups of 5 male and 5 female rats were exposed nose-only to target concentrations of 0 (control), 15, 45 or 135 mg/m3 N-methylaniline_EC 202-870-9 for 6 hours per day, 5 days per week during a 30-day period, with a total number of 20 exposure days. The concentration levels used were chosen in consultation with the sponsor on the basis of earlier studies with N-methylaniline and aniline (Treon et al., 1950; Pauluhn, 2004) and a limited pilot study to establish the concentrations at which cyanosis and increased methaemoglobin levels were

seen. To examine the toxicity of the test material, data on clinical observations, body weight gain, food consumption, haematology and clinical chemistry were used. In addition, animals were examined grossly at necropsy, organs were weighed, and a selection of organs and tissues (including the complete respiratory tract and nasal turbinates) was examined microscopically.

The mean actual concentrations (± standard deviation) of N-methylaniline_EC 202- 870-9 in the test atmospheres, based on HPLC analyses, were 13.3 (± 0,75), 40,0 (± 2.8), and 115.9 (± 7.1) mg/m3 for the low, mid and high concentration levels,

respectively.

Daily observation of the animals in the morning before the start of the exposures did not reveal treatment-related clinical abnormalities. At the group-wise observation during exposure, a decreased breathing frequency occasionally was seen in the

animals of the mid concentration group (on two occasions) and the animals of the high concentration group (three occasions). Additionally, two females of the midconcentration group showed cyanosis just after exposure one day in the third week

of the study. Treatment-related differences in body weight gain or food consumption were not seen.

Except prothrombin time, all red blood cell parameters showed changes in animals exposed to N-methylaniline_EC 202-870-9. For males exposed to the lowest concentration, haemoglobin and packed cell volume concentrations were decreased and the relative number of reticulocytes was increased. For female animals exposed to the lowest concentration red blood cell, haemoglobin and

packed cell volume were decreased and mean corpuscular volume and the percentage of reticulocytes were increased.

Effects on white blood cells were limited to an increase in white blood cells, lymphocytes, basophils and monocytes in female animals of the high concentration group.

Bilirubin was significantly increased in a concentration related manner in all exposed male groups and in females of the high concentration group. Creatinine was significantly decreased in female animals of the high concentration group.

Other treatment-related changes in clinical chemistry parameters were not seen.