4-methylmorpholine

Endpoint summary

Description of key information

Repeated dose toxicity - oral :

A reliable study with the related source substance NEM is available (Klimisch 2, according to OECD guideline 407, GLP). A NOAEL of 200 mg/kg bw/d was derived.

Repeated dose toxicity - inhalation :

One reliable study is available for the inhalation route (Klimisch 2 study). The study (DiPasquale LC, 1979) performed a subacute test according to a method similar or equivalent to OECD guideline 412 (GLP) in which rats were exposed to 100 or 1000 ppm test substance for two weeks. A NOAEC of 100 ppm or 414 mg/m³ air was derived.

 

Repeated dose toxicity - dermal :

No reliable studies were available for this route of exposure. However, no further testing is needed since reliable studies are available for repeated toxicity via the oral and inhalatory route (REACH regulation, Column 2 Adaptation, Annex VIII).  

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

read-across from related substance

Justification for type of information:

Read-across from structural analogue (4-ethylmorpholine). Justification of the read-across approach is included in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Based on reliable read-across information of N-ethylmorpholine is available (Klimisch 2), no adverse effect is expected. NOAEL is based on toxicologically adverse effects at 800 mg/kg bw/d only.

Critical effects observed:

no

System:

other: Based on reliable read-across information of N-ethylmorpholine is available (Klimisch 2), no adverse effect is expected.

Conclusions:

No reliable repeated dose toxicity study is available with the test substance. Data generated with the related substance 4-ethylmorpholine is used for endpoint coverage. The NOAEL for oral repeated dose toxicity in Cjr:CD(SD)IGS rats is considered to be 50 mg/kg bw/day for both sexes based on cage licking and chewing at 200 and 800 mg/kg bw/d. These clinical signs do not represent a toxicologically adverse effect and are furthermore not mentioned in an OECD421 study, performed in the same lab with the same rat strain. As relevant adverse effects are observed at 800 mg/kg bw/d only, a NOAEL of 200 mg/kg bw/d is proposed.
The same is assumed to be correct for the target substance.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2003 - 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Read-across GLP study performed according to OECD Guideline 407. The original study report is in Japanese language, although the figures and tables are in English. An English summary is available from the Japanese authorities and an extensive summary is present in the OECD HPV program files. After assessing the tables with results, the derived NOAEL seems to be underestimated.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): N-ethylmorpholine
- Molecular formula (if other than submission substance): C6 H13 N O
- Molecular weight (if other than submission substance): 115.18
- Smiles notation (if other than submission substance): CCN1CCOCC1
- InChl (if other than submission substance): InChI=1/C6H13NO/c1-2-7-3-5-8-6-4-7/h2-6H2,1H3
- Analytical purity: equal or more than 99%
- Impurities (identity and concentrations): 0.05% as moisture
- Lot/batch No.: 2901P0
- Stability under test conditions: The test solution was prepared and diluted to dosing concentrations by injection solvent every week. The diluted solution was confirmed to be stable for 8 days.
- Storage condition of test material: The test solution was kept in a refrigerator

Species:

rat

Strain:

other: Crj:CD(SD)IGS

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: 4 weeks old
- Weight at study initiation: day 1 mean weight: males 148.3 g (142.8-156.5 g) and females 126.9 g (120.3-133.7 g)
- Fasting period before study: unknown
- Acclimation period: 8 days before use

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test solution was prepared and diluted to dosing concentrations by injection solvent every week. They were kept in a refrigerator.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

GC method. Further details unknown.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

actual ingested

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

actual ingested

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

actual ingested

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

actual ingested

No. of animals per sex per dose:

10 at 0 and 800 mg/kg bw/day)
5 at 50 and 200 mg/kg bw/day)

Control animals:

yes, concurrent vehicle

Details on study design:

Recovery group: 0 (vehicle), 800 mg/kg bw/day
Recovery period: 14 days

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, daily

DETAILED CLINICAL OBSERVATIONS: Yes, once a week
Functional observations (ambulation, rearing, auditory, visual, proprioceptive stimuli, grip strength) in 4th week of dosing period and 2nd week of recovery period.

BODY WEIGHT: Yes (incl. body weight changes and body weight gain)
On day 1, 2, 4, 8, 11, 15, 18, 22, 25, 28 of dosing period and on day 1, 4, 8, 11, 14 of recovery period

FOOD CONSUMPTION: Yes
On day 1, 8, 15, 22 of dosing period and on day 1 and 8 of recovery period.

HAEMATOLOGY: Yes, at end of dosing period and at the end of the recovery period.
Parameters examined: RBC, hemoglobin, hematocrit, MCV, MCH, MCHC, platelet, PT, APTT, WBC, % of neutrophils, eosinophils, basophils, monocytes, lymphocytes

CLINICAL CHEMISTRY: Yes, at the end of dosing period and at the end of the recovery period.
Parameters examined: total protein, albumin, A/G, BUN, creatinine, glucose, total cholesterol, triglyceride, total bilirubin, inorganic P, Ca, Na, K, Cl, ALP, GPT, GOT, gamma-GTP

URINALYSIS: Yes, on day 23 of dosing period .
Parameters examined: urinary volume, specific gravity, pH, color, turbidity, protein, glucose, ketone, bilirubine, occult blood, urobilinogen, sodium, potassium and chlorine.
Microscopic examination of urinary sediment: red blood cells, crystal, cast, white blood cells, epithelial cells.

ORGAN WEIGHT: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, at the end of dosing period and at the end of recovery period.
Absolute organ weights: brain, thymus, heart, liver, kidneys, spleen, adrenal glands, testes, epididymides, ovaries.

HISTOPATHOLOGY: Yes
Organs examined: liver, kidney, spleen, heart, prostate, ovary, lung & bronchus, adrenal gland, stomach, ileum, colon, mesenteric and submandibular lymphnodes, thymus, trachea, brain, spinal cord, sciatic nerve & gastrocnemial muscle, thyroid gland, testis, epididymis, uterus, urinary bladder, bone & marrow of femur, jejunum.

Statistics:

Barlett's tests were initially performed for body weight, food consumption, hematological examination results, hemostasis examination results, blood chemical examination results, urinalysis results (urine volume and urinary osmolality), organ weight, and relative organ weight. When their values were equal variance on Barlett's tests, Dunnett's multiple comparison tests were performed and the significant difference between dose groups were examined. Fischer's exact tests were performed for pathological examination. Additionally, F-test, Student t-test, Aspin-Welch t-test, chi-square test, Wilcoxon test, and mann-Whitney U-test were also used.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Both sexes:
In 200 or 800 mg/kg bw/day group: cage-licking and chewing were observed.
In 800 mg/kg bw/day group: Action tremors, decrease in movement, a crouching position, eyelid closure and salivation were observed. In detailed clinical observation, increased incidences of slight irritability, of touch responses vocalization and action tremors were observed. A few females showed intermitted walking and prone position. In the functional test at the 4th week of administration, lower number of rearing was observed in females during the first 30 minuts after administration. Numbers of animals showing no ambulation trend to decrease in both sexes during each testing period. In the recovery period, a high number of rearing was observed in males during the first 30 minutes of observation. No other clinical signs were observed during the recovery period.

Mortality:

mortality observed, treatment-related

Description (incidence):

- Both sexes: No deaths were observed in any of the treatment groups during dosing and recovery period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Male: The body weight gains at 800 mg/kg bw/day decreased significantly from day 2 of the administration period to the end of the recovery period.
- Female: The body weight gains at 800 mg/kg bw/day were lower from day 4 of the administration period to the end of recovery period, and decreased significantly from day 4 to 11, on day 28 of the administration period, and from day 4 to day 14 of the recovery period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Male: The food consumption at 800 mg/kg bw/day decreased significantly from day 1 of the administration period to day 1 of the recovery period.
- Female: The food consumption at 800 mg/kg bw/day decreased significantly from day 1 to 8 of the administration period.

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):

after administration period:
- Male: Reduction of prothrombin time and activated partial thromboplastin time were found at 800 mg/kg bw/day. Although significant reductions for PT were found at 50 and 200 mg/kg bw/day, the author judged that these changes had no toxicological meaning.
- Female: Although reduction of prothrombin time was found at 50 mg/kg bw/day, the author judged that these changes were non-toxicological. Higher percentage of neutrophil and monocyte and lower percentage of eosinophil and lymphocyte in differential leukocyte ratio, and increase in platelet were found at 800 mg/kg bw/day.

after recovery period:
- Male: None
- Female: Increase in platelet was found at 800 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

after administration period:
- Male:
in 800 mg/kg bw/day group: Increase in inorganic phosphate, calcium and blood urea nitrogen; decreases in chloride and albumin
- Female:
in 800 mg/kg bw/day group: Increase in inorganic phosphate, glucose and triglyceride; decreases in chloride, GOT and total bilirubin. GOT was also decreased in 50 and 200 mg/kg bw/d females.

after recovery period:
- Male:
in 800 mg/kg bw/day group: increases in A/G ratio
- Female:
in 800 mg/kg bw/day group: decreases in total protein, albumin, glucose.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

- Male: Protein was decreased in 800 mg/kg bw group
- Female: Keton bodies and urobilinogen was increased and specific gravity was decreased in 800 mg/kg bw group
No effects during recovery period were observed.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

after administration period:
- Male:
in 800 mg/kg bw/day group: decrease in absolute weight of epididymides, increase in relative weight of brain, liver, kidneys, adrenal glands and testes.
- Female:
in 800 mg/kg bw/day group: decrease in absolute weight of brain, increase in absolute weight of liver, increase in relative weights of liver and kidneys

after recovery period:
- Male:
in 800 mg/kg bw/day group: decrease in absolute weight of heart and liver, increase in relative weight of brain
- Female:
in 800 mg/kg bw/day group: increase in relative weight of brain

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopically: No toxicological change was observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

after administration period:
- Male:
in 800 mg/kg bw/day group:
Very slight hypertrophy of centrilobular hepatocytes in liver (1/5)
Very slight vacuolation of epithelium in distal and Henle's loop in kidneys (3/5)
- Female:
in 800 mg/kg bw/day group:
Very slight or slight hypertrophy of centrilobular hepatocytes in liver (4/5)
Very slight microgranuloma in liver (1/5)
Very slight and slight vacuolation of epithelium in distal and Henle's loop in kidneys (2/5)
(No. of animal with change/No. of animal tested)

After recovery period:
- Male: None
- Female: None

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Dose descriptor:

NOAEL

Remarks:

disregarded

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: This NOAEL was derived by the authors of the study, based on clinical signs (cage licking and chewing) at 200 and 800 mg/kg bw/d. As these clinical signs do not constitute an adverse effect, this NOAEL is disregarded.

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on toxicologically adverse effects at 800 mg/kg bw/d only.

Critical effects observed:

not specified

Table 1 Hematology of rats treated orally with
N-ethylmorpholine in the twenty-eight-day repeat dose
toxicity test
------------------------------------------------------------
                       After the        After the
                       administrationrecovery
                       period             period
Dose               0         800     0         800
                         (mg/kg b.w./day)
------------------------------------------------------------
Male
No. of animals5         5         5         5
------------------------------------------------------------
PT (sec)          22.6    13.6**16.6    17.4
 +/-S.D.          3.1      1.3      2.3      2.6
APTT (sec)     24.3    19.8**24.4    25.0
 +/-S.D.          2.1      1.0      1.9      1.1
------------------------------------------------------------
Female
No. of animals5         5         5         5
------------------------------------------------------------
Platelet (X10E4/uL)
                       100.4  115.3*97.0    112.8*
 +/-S.D.          12.3    7.4      11.0    8.6
Neutrophil (%)           6         12*     9         8
 +/-S.D.          2         5         3         3
Eosinophil (%)           2         0**     2         2
 +/-S.D.          1         0         1         0
Monocyte (%)2         5*       3         3
 +/-S.D.          1         2         0         2
Lymphocyte (%)
                       90       83       86       87
 +/-S.D.          3         6         3         4
------------------------------------------------------------
PT: prothrombin time, APTT; activated partial thromboplastin
time
Significant difference from control group; *p=<0.05,
**p=<0.01

Table 2 Blood chemical examination of rats treated orally
with N-ethylmorpholine in the twenty-eight-day repeat dose
toxicity test
------------------------------------------------------------
                       After the        After the
                       administrationrecovery
                       period             period
Dose               0         800     0         800
                         (mg/kg b.w./day)
------------------------------------------------------------
Male
No. of animals5         5         5         5
------------------------------------------------------------
Albumin (g/dL)          2.9      2.6*    3.0      3.1
 +/-S.D.          0.1      0.2      0.0      0.1
A/G                1.28    1.20    1.21    1.42*
 +/-S.D.          0.13    0.08    0.08    0.16
BUN (mg/dL)14       19*     18       17
 +/-S.D.          1         2         2         1
Inorganic phosphorus (mg/dL)
                       8.1      9.9*    7.9      7.8
 +/-S.D.          0.2      0.9      0.3      0.3
Ca (mg/dL)    8.9      9.5*    9.2      9.1
 +/-S.D.          0.2      0.4      0.2      0.2
Cl (mEq/dL)   107.5  104.5*105.9  107.7
 +/-S.D.          1.0      1.9      0.6      1.6
------------------------------------------------------------
Female
No. of animals5         5         5         5
------------------------------------------------------------
Total protein (g/dL)
                       4.9      4.7      5.7      5.4*
 +/-S.D.          0.2      0.2      01.      0.1
Albumin (g/dL)          2.9      2.7      3.5      3.3*
 +/-S.D.          0.2      0.2      0.1      0.1
Glucose (mg/dL)
                       100     124** 131     104*
 +/-S.D.          7         12       21       12
Triglyceride (mg/dL)
                       10       28*     14       14
 +/-S.D.          3         19       4         6
Total bilirubin (mg/dL)
                       0.10    0.05**0.12    0.10
 +/-S.D.          0.02    0.02    0.02    0.03
Inorganic phosphorus (mg/dL)
                       7.2      8.6**  6.4      6.4
 +/-S.D.          0.5      0.6      0.7      0.8
Cl (mEq/dL)   110.4  106.4*107.5  109.0
 +/-S.D.          1.2      0.5      0.9      1.8
------------------------------------------------------------
Significant difference from control group; *p=<0.05,
**p=<0.01

Table 3 Absolute and relative organ weights of rats treated
orally with N-ethylmorpholine in the twenty-eight day repeat
dose toxicity test
------------------------------------------------------------
                       After the        After the
                       administrationrecovery
                       period             period
Dose               0         800     0         800
                         (mg/kg b.w./day)
------------------------------------------------------------
Male
No. of animals5         5         5         5
------------------------------------------------------------
Heart              1210.4992.0  1472.11164.1**
 +/-S.D.          45.4    104.6  141.2  118.1
Liver               11721 10504 13403 11036*
 +/-S.D.          573.8  1362.51205.81316.9
Epididymides 623.1  552.9*973.3  933.6
 +/-S.D.          26.0    34.5    90.8    72.9
-Relative organ weight (mg/g)
Brain              5.585  6.754*4.792  5.504*
 +/-S.D.         0.307  0.482  0.352  0.584
Liver              33.36  38.17**          31.05  30.54
 +/-S.D.          1.22    3.56    2.32    1.57
Kidneys          7.91    9.90**7.32    7.88
 +/-S.D.          0.52    0.90    0.60    0.51
Adrenal glands           0.15    0.22**0.15    0.17
 +/-S.D.          0.01    0.04    0.03    0.02
------------------------------------------------------------
Female
No. of animals5         5         5         5
------------------------------------------------------------
-Absolute organ weight (g)
Brain              1794.31687.5*          1823.71819.1
 +/-S.D.         98.8    47.3    65.2    17.4
Liver              6530   8353**           7974   7002
 +/-S.D.          739     1026   1086   808
-Relative organ weight (mg/g)
Brain              9.024  8.677  7.121  8.126*
 +/-S.D.         0.835  0.826  0.598  0.381
Liver              32.64  42.61**          30.88  31.26
 +/-S.D.          1.65    2.69    1.68    3.74
Kidneys          9.16    10.79**          8.04    8.50
 +/-S.D.          0.61    0.72    0.32    0.80
------------------------------------------------------------
Significant difference from control group; *p=<0.05,
**p=<0.01

Table 4 Summary of histopathological findings of rats
treated orally with N-ethylmorpholine in the
twenty-eight-day repeat dose toxicity test
------------------------------------------------------------
                       After the        After the
                       administrationrecovery
                       period             period
Dose               0         800     0         800
                         (mg/kg b.w./day)
------------------------------------------------------------
Male
No. of animals5         5         5         5
------------------------------------------------------------
-Liver
Hypertrophy of centrilobular hepatocytes
                       0         1         0         0
-Kidneys
Vacuolation of epithelium in distal and Henle's loop
                       0         3         0         0
------------------------------------------------------------
Female
No. of animals5         5         5         5
------------------------------------------------------------
-Liver
Hypertrophy of centrilobular hepatocytes
                       0         4*       0         0
Microgranuloma
                       0         1         0         2
-Kidneys
Vacuolation of epithelium in distal and Henle's loop
                       0         2         0         0
------------------------------------------------------------
Significant difference from control group; *p=<0.05,
**p=<0.01

Conclusions:

The NOAEL for oral repeated dose toxicity in Cjr:CD(SD)IGS rats is considered to be 50 mg/kg bw/day for both sexes based on cage licking and chewing at 200 and 800 mg/kg bw/d. These clinical signs do not represent a toxicologically adverse effect and are furthermore not mentioned in an OECD 421 study, performed in the same lab with the same rat strain. As relevant adverse effects are observed at 800 mg/kg bw/d only, a NOAEL of 200 mg/kg bw/d is proposed.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979-04-25 - 1979-05-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

deviations: only 2 concentrations tested instead of 3. Raw data and tables were not available.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

only 2 concentrations tested instead of 3

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): N-methylmorpholine (NMM)
- Substance type: Volatile colorless liquid
- Physical state: liquid
- Purity: no data
- Lot/batch No.: 85-309
- Other: vapor pressure: 18 mm of mercury at 20°C and a distinctive amine-type, fishy odor

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Holtzman Co., Madison, Wisconsin, USA
- Age at study initiation: young adult rats
- Weight at study initiation: 187-330 g (no data on mean weights per sex available)
- Housing: Animals were individually caged in ten-compartment wire mesh cages with food available during all non-exposure periods.
- Diet (e.g. ad libitum): Charles River Rat/Mouse/Hamster Diet
- Water (e.g. ad libitum): Water was available ad libitum with the exception of the one hour exposure during which exposed and control groups were without water.
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
During non-exposure periods the animals were maintained in a controlled environment:
- Temperature (°C): 21°C -26°C
- Humidity (%): 40-80% relative humdidity
- Photoperiod (hrs dark / hrs light): 12 hour light-dark cycle

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: not applicable (vapour)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Inhalation exposures were conducted under dynamic conditions using three 1000 L glass and stainless steel inhalation chambers with filtered room air as the diluent. Chambers were operated at an airflow of approximately 350 (275-370) liters per minute and kept at a slight negative pressure to prevent outward leakage of the test material. Under these chanmber volume and airflow conditions the theoretical chamber volume and airflow conditions the theoretical chamber build-up/clearance time (T99) is 13.1 minutes. Temperature and relative humidity in the chambers were maintained at 24°C and 55 ± 10% respectively.
Vapor concentrations of NMM were generated by infusing the liquid material at a constant rate, via syringe pumps (Razel Model A-99 for 100 ppm NMM; Sage Model 220 for 1000 ppm NMM) into glass round bottom flasks placed in heating jackets. Generation flasks were heated to enhance volatilization of the NMM. A pre-filtered, regulated airflow of 10 liters per minute was passed through each flask to carry the vapors into the chambers. Each vapor generation system was positioned in a glass and stainless steel ante-chamber located at the top inlet of the exposure chamber. Removal of the 1 hour groups (Air Control, 100 ppm NMM, 1000 ppm NMM) at the conclusion of each exposure was facilitated by using hinged panels on the chamber doors. This procedure permitted rapid removal of animal exposure cages while minimizing the loss of chamber atmospheric concentrations. Upon removal from the chamber, one hour NMM exposure groups were placed in a fume hood for 15 minutes before being observed and returned to the animal room.
After completion of each daily, 7 hour exposure, NMM vapor generation systems were shut down and all exposure chambers purged with air for 30 minutes before removing rats for observation and movement to the animal room. Chambers were then sprayed with water to remove any residual NMM, feces or urine.
A positive pressure, full-face respirator connected to a clean air breathing supply was worn whenever personnel filled or adjusted vapor generation equipment or removed NMM-exposed animals from the exposure chambers.

TEST ATMOSPHERE
Chamber concentrations of NMM were monitored using a H.N.U. Systems photoionization detector, factory-calibrated for NMM with isobutylene as a reference standard. The analytical instrument was time-shared between the two NMM exposure chambers by using a switching valve and alternating sample source every 15 minutes or as desired. The photoionization detector was calibrated for NMM at least twice daily (before initiation and at the conclusion of the 7 hr exposure) using known concentrations of isobutylene in air. Chamber airflow was set each morning before exposures began using a Hastings Raydist Precision Air Meter. Chamber temperature and relative humidity were recorded hourly from an Abbeon Certified hygrometer and temperature indicator.

VEHICLE (if applicable)
Filtered air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Daily and ten-day mean NMM chamber concentrations and standard deviations are presented. Because the analytical instrument was time-shared, daily means for one hour exposures were derived from chamber analyses recorded every 5 minutes while seven-hour means represent chamber concentrations recorded at 30 minute intervals. The mean ten-day NMM concentrations were 102.6 ± 9.3 ppm (1 hour), 103.9 ± 11.4 ppm (7 hours), 997.4 ± 50.2 ppm (1 hour) and 1005.3 ± 103.4 ppm (7 hours).

Duration of treatment / exposure:

1 or 7 hours

Frequency of treatment:

five days a week for two weeks

Dose / conc.:

100 ppm (nominal)

Remarks:

= 414 mg/m³

Dose / conc.:

1 000 ppm (nominal)

Remarks:

= 4140 mg/m³

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

No data
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): rats were grouped after stratifying body weights into 6 strat and subsequent random allocation to exposure gorups.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- All rats were closely observed for any unusual behavioral trends, pharmatoxic signs, morbidity or mortality. Daily observations were made before, during and after each exposure. Any abnormal findings were recorded on an animal observation sheet.

BODY WEIGHT: Yes
- Individual body weights were obtained twice before the start of the exposure (at -11 and -4 days), and at 0, 7, 14 and 28 days. All body weights were recorded on a body weight data sheet.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Individual blood samples were drawn from the abdominal aorta of 5 male and 5 female rats from each group during serial sacrifices which occurred on day 0, and 14 and 28 days after first exposure. Animals were anesthetized with Ketaset Plus before samples were taken. A computer generated table of random numbers was used to control for bias in the selection of animals for serial sacrifice.
The following hematological measurements were determined in-house using the methods described in the GMEL Standard Operating Procedures for Clinical Chemistry Evaluations (SOP-CC):
Hemoglobin, Hematocrit, Red Blood Cell, White Blood Cell, Platelets, Differential, Reticulocyte, Methemoglobin, Mean Red Blood Cell Fragility, Mean Corpuscular Volume

CLINICAL CHEMISTRY: Yes
Serum and bone marrow samples were delivered to the Bionetics Medical Laboratories for determination of the following serum chemistry and cytologic measurements:
Glucose, Blood Urea Nitrogen, Creatinine, Uric Acid, Cholesterol, Calcium, Phosphorus, Total Protein, Albumin, Globulin, Bilirubin, Alkaline Phosphatase, Lactic Dehydrogenase, Serum Glutamic Oxaloacetic Transaminase, Bone Marrow Differential Count

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross pathologic examinations were conducted on all animals at the time of sacrifice. Organ weights were obtained for the liver, kidney, spleen, brain and lungs with trachea. These same organs and tissues were preserved in 10% neutral buffered formalin and processed for subsequent histopathological examination by a Board Certified Veterinary Pathologist.
HISTOPATHOLOGY: Yes
Paraffin-embedded sections were cut on an American Optical Model 820 rotary microtome at a thickness of 4 µ and stained with hematoxylin and eosin.

Statistics:

Statistical tests using analysis of variance (ANOVA) were made for the following: hematology data, serum chemistry data, body weight at necropsy, organ weight at necropsy, organ/body weight, organ/brain weight, bone marrow data.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Exposure related signs, however, were noted to occur in all test groups during the exposure sequence. Nasal discharge was observed periodically in five male and six female rats from the one hour 1000 ppm NMM exposure groups, and six male and six female rats from the seven hour 1000 ppm NMM exposure groups. Although slight nasal discharge was seen in all groups including controls, the frequency of occurrence was increased in rats exposed to 1000 ppm NMM.
Salivation was observed periodically in one male and five female rats from the one hour 1000 ppm test item-exposure groups, and four male, and two female rats from the seven hour 1000 ppm test item-exposure groups. With the exception of four females from the one hour group, salivation was noted to occur most freqently during the second week of exposure. Salivation was not observed in either the control or 100 ppm test item-exposure groups.
Lacrimation was seen on day 3 in a male rat from one hour 100 ppm test item group and periodically in three female rats from the on hour 1000 ppm test item group. On the third day of exposure, a female rat exhibited tremors, excessive masticatory and spastic preening movements followed by slightly depressed, labored respiration. These signs disappeared, however, by the following day.
Three rats exhibited persistent eye inflammation or injury not related to exposure to the test material (7 hr control male, 7 hr control female, 7 hr 1000 ppm female). A one hour male control had a porphyrin-like encrustation around the eyes on day 2.
Transient (less than 24 hours), diffuse clouding of either the right, left or both eyes during and following exposure was found in one male and three female rats exposed to 100 ppm test item for one hour, two male rats exposed to 100 ppm test item for 7 hours, and all forty rats exposed to 1000 ppm test item for either one or seven hours. The times of onset of this response were approximately: day 2 for the 100 ppm tet item one hour exposure; day 7 for the 100 ppm test item seven hour exposure; day 10 for the 1000 ppm test item one hour exposure; and day 8 for the 1000 ppm test item seven hour exposure. Pie-shaped areas of apparent corneal opacity were also noted in two of forty control rats, four of forty 100 ppm test item-exposed rats, and six of forty 1000 ppm test item-exposed rats. This ocular abnormality, however, is not believed to be due to exposure to the test material, whereas the transient clouding does not appear to be associated with test item inhalation. No current explanation can be offered as to the potential biological significance of this finding.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no unscheduled deaths during the exposure and subsequent two-week holding period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Growth curves exhibited by rats in all groups were considered to be normal over the 28-day test and holding period. male rats exposed to 1000 ppm test substance for either one hour or seven hours showed slightly depressed weight gains during the exposure sequence followed by a rebound during the two-week holding period in the seven hour group. The lack of rebound in the one hour group, however, is unexplained. Although slight differences in group mean body weights were noted at necropsy (Day 28), none were found to be statistically significant.

Food consumption and compound intake (if feeding study):

not examined

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):

Although several findings of statistical significance were noted to occur, the only contrast of possible biological significance was the increase over time in the percent of atypical or immature lymphocytes noted to occur at day 14 for both male and female rats from 100 and 1000 ppm test item treatment groups and at day 28 in the 1000 ppm test item-exposed male rats. Although the atypical lymphocyte data is only marginally quantitative (i.e. data is expressed as a percentage of 100 cells counted and the total numbers found were small, ranging from 1 to 7%) and no treatment-related statistically significant differences were noted to occur in the lymphocytic series in the bone marrow, the present finding cannot be dismissed as a spurious significant contrast. Additionally, a concommitant increase in the lymphocyte differential cell and white blood cell counts should accompany any substantial increase in atypical lymphocytes. A statistically significant increase in the lymphocyte differential count was not seen, however, but the total WBC count was elevated significantly at day 14 for rats exposed to 1000 ppm test substance. Although elevated, the WBC counts were within the range of values exhibited by all other groups over the course of the study. The possibility exists that the atypical lymphocyte increase may represent the genesis of some abnormality. Any possible biological significance of this finding, however, cannot be assessed at this time.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

- Serum Chemistry and Enzymes:
Although several findings of statistical significance were noted to occur, none were judged to be related to exposure to the test substance.
- Bone marrow differential counts:
An examination of the data did not reveal any abnormalities.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Although several statistically significant contrasts were noted to occur in the organ weight data, none were judged to be related to exposure to the test substance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross pathologic examinations conducted on animals at day 0 and 14 and 28 days after the first exposure did not reveal any consistent abnormal findings that could be attributed to inhalation exposure to the test substance. One control rat, killed at day 0, had a blanched and frim caudate lobe of the liver. Another day 0 control had pitted renal surfaces. The day 28 examination revealed pitted kidneys in two controls; and eroded renal medulla in a seven hour test item-exposed rat; and an enlarged, thick, firm red-streaked area on the right lobe of the liver of a control rat. The most prevalent finding, common to both control and test item-exposed rats and seen at all examination intervals, was pulmonary in origin and consisted of consolidated areas, small watery-like cysts and shine pinpoint spots on various lobes of the lung.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Chronic respiratory disease was the most prevalent finding and was seen with equal frequency and severity in both control and test item-exposed animals. The presence of hepatic microgranulomata was also noted in control rats and rats exposed to the test substance. All lesions noted microscopically were considered the result of intercurrent disease processes and not as the result of exposure to the test substance.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEC

Effect level:

100 ppm

Based on:

test mat.

Remarks:

= 414 mg/m³

Sex:

male/female

Basis for effect level:

other: Elevations in atypical lymphocyte differentials at day 14 in female rats exposed to 1000 ppm test substance for 7 hours a day

Critical effects observed:

not specified

Lymphocytes

To further clarify the increase in atypical lymphocytes, a recount was performed in which 500 white blood cells were counted and classified by 2 independent observers. Given the agreement between the independent 500 cell recounts, it seems clear that the level of plasmoid lymphocytes was high on day 14 for the 1000 ppm test item 7 hour exposed females. This is in contrast to the less quantitative, original 100 cell differential results which indicated elevations at day 14 for both male and female rats from 100 and 1000 ppm test item groups and at day 28 in the 1000 ppm test item males. In humans, the plasmoid lymphocyte or Turk cell is clinically significant in individuals with viral, parasitic, mycotic or bacterial infections as well as hypersensitive, drug or chemical toxic states. Based upon the information at hand, it is difficult to make a conclusion with respect to the role of test item in producing the changes seen in rats from the present study.

 

Dose levels: conversion from ppm to mg/m³

The following formula was used to convert the dose level in ppm to mg/m³:

dose in mg/m³ = [(gram molecular weight of substance) x (dose in ppm)]/24.45

(reference on site at the sponsor)

Conclusions:

The results of the current study suggest that gross signs of irritation, transient cloudiness of the eye, and possibly elevations in atypical lymphocyte differential counts were the only detectable manifestations of exposure of rats to test substance vapors for five days per week for two weeks.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

414 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979-04-25 - 1979-05-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

deviations: only 2 concentrations tested instead of 3. Raw data and tables were not available.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

only 2 concentrations tested instead of 3

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): N-methylmorpholine (NMM)
- Substance type: Volatile colorless liquid
- Physical state: liquid
- Purity: no data
- Lot/batch No.: 85-309
- Other: vapor pressure: 18 mm of mercury at 20°C and a distinctive amine-type, fishy odor

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Holtzman Co., Madison, Wisconsin, USA
- Age at study initiation: young adult rats
- Weight at study initiation: 187-330 g (no data on mean weights per sex available)
- Housing: Animals were individually caged in ten-compartment wire mesh cages with food available during all non-exposure periods.
- Diet (e.g. ad libitum): Charles River Rat/Mouse/Hamster Diet
- Water (e.g. ad libitum): Water was available ad libitum with the exception of the one hour exposure during which exposed and control groups were without water.
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
During non-exposure periods the animals were maintained in a controlled environment:
- Temperature (°C): 21°C -26°C
- Humidity (%): 40-80% relative humdidity
- Photoperiod (hrs dark / hrs light): 12 hour light-dark cycle

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: not applicable (vapour)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Inhalation exposures were conducted under dynamic conditions using three 1000 L glass and stainless steel inhalation chambers with filtered room air as the diluent. Chambers were operated at an airflow of approximately 350 (275-370) liters per minute and kept at a slight negative pressure to prevent outward leakage of the test material. Under these chanmber volume and airflow conditions the theoretical chamber volume and airflow conditions the theoretical chamber build-up/clearance time (T99) is 13.1 minutes. Temperature and relative humidity in the chambers were maintained at 24°C and 55 ± 10% respectively.
Vapor concentrations of NMM were generated by infusing the liquid material at a constant rate, via syringe pumps (Razel Model A-99 for 100 ppm NMM; Sage Model 220 for 1000 ppm NMM) into glass round bottom flasks placed in heating jackets. Generation flasks were heated to enhance volatilization of the NMM. A pre-filtered, regulated airflow of 10 liters per minute was passed through each flask to carry the vapors into the chambers. Each vapor generation system was positioned in a glass and stainless steel ante-chamber located at the top inlet of the exposure chamber. Removal of the 1 hour groups (Air Control, 100 ppm NMM, 1000 ppm NMM) at the conclusion of each exposure was facilitated by using hinged panels on the chamber doors. This procedure permitted rapid removal of animal exposure cages while minimizing the loss of chamber atmospheric concentrations. Upon removal from the chamber, one hour NMM exposure groups were placed in a fume hood for 15 minutes before being observed and returned to the animal room.
After completion of each daily, 7 hour exposure, NMM vapor generation systems were shut down and all exposure chambers purged with air for 30 minutes before removing rats for observation and movement to the animal room. Chambers were then sprayed with water to remove any residual NMM, feces or urine.
A positive pressure, full-face respirator connected to a clean air breathing supply was worn whenever personnel filled or adjusted vapor generation equipment or removed NMM-exposed animals from the exposure chambers.

TEST ATMOSPHERE
Chamber concentrations of NMM were monitored using a H.N.U. Systems photoionization detector, factory-calibrated for NMM with isobutylene as a reference standard. The analytical instrument was time-shared between the two NMM exposure chambers by using a switching valve and alternating sample source every 15 minutes or as desired. The photoionization detector was calibrated for NMM at least twice daily (before initiation and at the conclusion of the 7 hr exposure) using known concentrations of isobutylene in air. Chamber airflow was set each morning before exposures began using a Hastings Raydist Precision Air Meter. Chamber temperature and relative humidity were recorded hourly from an Abbeon Certified hygrometer and temperature indicator.

VEHICLE (if applicable)
Filtered air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Daily and ten-day mean NMM chamber concentrations and standard deviations are presented. Because the analytical instrument was time-shared, daily means for one hour exposures were derived from chamber analyses recorded every 5 minutes while seven-hour means represent chamber concentrations recorded at 30 minute intervals. The mean ten-day NMM concentrations were 102.6 ± 9.3 ppm (1 hour), 103.9 ± 11.4 ppm (7 hours), 997.4 ± 50.2 ppm (1 hour) and 1005.3 ± 103.4 ppm (7 hours).

Duration of treatment / exposure:

1 or 7 hours

Frequency of treatment:

five days a week for two weeks

Dose / conc.:

100 ppm (nominal)

Remarks:

= 414 mg/m³

Dose / conc.:

1 000 ppm (nominal)

Remarks:

= 4140 mg/m³

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

No data
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): rats were grouped after stratifying body weights into 6 strat and subsequent random allocation to exposure gorups.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- All rats were closely observed for any unusual behavioral trends, pharmatoxic signs, morbidity or mortality. Daily observations were made before, during and after each exposure. Any abnormal findings were recorded on an animal observation sheet.

BODY WEIGHT: Yes
- Individual body weights were obtained twice before the start of the exposure (at -11 and -4 days), and at 0, 7, 14 and 28 days. All body weights were recorded on a body weight data sheet.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Individual blood samples were drawn from the abdominal aorta of 5 male and 5 female rats from each group during serial sacrifices which occurred on day 0, and 14 and 28 days after first exposure. Animals were anesthetized with Ketaset Plus before samples were taken. A computer generated table of random numbers was used to control for bias in the selection of animals for serial sacrifice.
The following hematological measurements were determined in-house using the methods described in the GMEL Standard Operating Procedures for Clinical Chemistry Evaluations (SOP-CC):
Hemoglobin, Hematocrit, Red Blood Cell, White Blood Cell, Platelets, Differential, Reticulocyte, Methemoglobin, Mean Red Blood Cell Fragility, Mean Corpuscular Volume

CLINICAL CHEMISTRY: Yes
Serum and bone marrow samples were delivered to the Bionetics Medical Laboratories for determination of the following serum chemistry and cytologic measurements:
Glucose, Blood Urea Nitrogen, Creatinine, Uric Acid, Cholesterol, Calcium, Phosphorus, Total Protein, Albumin, Globulin, Bilirubin, Alkaline Phosphatase, Lactic Dehydrogenase, Serum Glutamic Oxaloacetic Transaminase, Bone Marrow Differential Count

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross pathologic examinations were conducted on all animals at the time of sacrifice. Organ weights were obtained for the liver, kidney, spleen, brain and lungs with trachea. These same organs and tissues were preserved in 10% neutral buffered formalin and processed for subsequent histopathological examination by a Board Certified Veterinary Pathologist.
HISTOPATHOLOGY: Yes
Paraffin-embedded sections were cut on an American Optical Model 820 rotary microtome at a thickness of 4 µ and stained with hematoxylin and eosin.

Statistics:

Statistical tests using analysis of variance (ANOVA) were made for the following: hematology data, serum chemistry data, body weight at necropsy, organ weight at necropsy, organ/body weight, organ/brain weight, bone marrow data.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Exposure related signs, however, were noted to occur in all test groups during the exposure sequence. Nasal discharge was observed periodically in five male and six female rats from the one hour 1000 ppm NMM exposure groups, and six male and six female rats from the seven hour 1000 ppm NMM exposure groups. Although slight nasal discharge was seen in all groups including controls, the frequency of occurrence was increased in rats exposed to 1000 ppm NMM.
Salivation was observed periodically in one male and five female rats from the one hour 1000 ppm test item-exposure groups, and four male, and two female rats from the seven hour 1000 ppm test item-exposure groups. With the exception of four females from the one hour group, salivation was noted to occur most freqently during the second week of exposure. Salivation was not observed in either the control or 100 ppm test item-exposure groups.
Lacrimation was seen on day 3 in a male rat from one hour 100 ppm test item group and periodically in three female rats from the on hour 1000 ppm test item group. On the third day of exposure, a female rat exhibited tremors, excessive masticatory and spastic preening movements followed by slightly depressed, labored respiration. These signs disappeared, however, by the following day.
Three rats exhibited persistent eye inflammation or injury not related to exposure to the test material (7 hr control male, 7 hr control female, 7 hr 1000 ppm female). A one hour male control had a porphyrin-like encrustation around the eyes on day 2.
Transient (less than 24 hours), diffuse clouding of either the right, left or both eyes during and following exposure was found in one male and three female rats exposed to 100 ppm test item for one hour, two male rats exposed to 100 ppm test item for 7 hours, and all forty rats exposed to 1000 ppm test item for either one or seven hours. The times of onset of this response were approximately: day 2 for the 100 ppm tet item one hour exposure; day 7 for the 100 ppm test item seven hour exposure; day 10 for the 1000 ppm test item one hour exposure; and day 8 for the 1000 ppm test item seven hour exposure. Pie-shaped areas of apparent corneal opacity were also noted in two of forty control rats, four of forty 100 ppm test item-exposed rats, and six of forty 1000 ppm test item-exposed rats. This ocular abnormality, however, is not believed to be due to exposure to the test material, whereas the transient clouding does not appear to be associated with test item inhalation. No current explanation can be offered as to the potential biological significance of this finding.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no unscheduled deaths during the exposure and subsequent two-week holding period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Growth curves exhibited by rats in all groups were considered to be normal over the 28-day test and holding period. male rats exposed to 1000 ppm test substance for either one hour or seven hours showed slightly depressed weight gains during the exposure sequence followed by a rebound during the two-week holding period in the seven hour group. The lack of rebound in the one hour group, however, is unexplained. Although slight differences in group mean body weights were noted at necropsy (Day 28), none were found to be statistically significant.

Food consumption and compound intake (if feeding study):

not examined

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):

Although several findings of statistical significance were noted to occur, the only contrast of possible biological significance was the increase over time in the percent of atypical or immature lymphocytes noted to occur at day 14 for both male and female rats from 100 and 1000 ppm test item treatment groups and at day 28 in the 1000 ppm test item-exposed male rats. Although the atypical lymphocyte data is only marginally quantitative (i.e. data is expressed as a percentage of 100 cells counted and the total numbers found were small, ranging from 1 to 7%) and no treatment-related statistically significant differences were noted to occur in the lymphocytic series in the bone marrow, the present finding cannot be dismissed as a spurious significant contrast. Additionally, a concommitant increase in the lymphocyte differential cell and white blood cell counts should accompany any substantial increase in atypical lymphocytes. A statistically significant increase in the lymphocyte differential count was not seen, however, but the total WBC count was elevated significantly at day 14 for rats exposed to 1000 ppm test substance. Although elevated, the WBC counts were within the range of values exhibited by all other groups over the course of the study. The possibility exists that the atypical lymphocyte increase may represent the genesis of some abnormality. Any possible biological significance of this finding, however, cannot be assessed at this time.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

- Serum Chemistry and Enzymes:
Although several findings of statistical significance were noted to occur, none were judged to be related to exposure to the test substance.
- Bone marrow differential counts:
An examination of the data did not reveal any abnormalities.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Although several statistically significant contrasts were noted to occur in the organ weight data, none were judged to be related to exposure to the test substance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross pathologic examinations conducted on animals at day 0 and 14 and 28 days after the first exposure did not reveal any consistent abnormal findings that could be attributed to inhalation exposure to the test substance. One control rat, killed at day 0, had a blanched and frim caudate lobe of the liver. Another day 0 control had pitted renal surfaces. The day 28 examination revealed pitted kidneys in two controls; and eroded renal medulla in a seven hour test item-exposed rat; and an enlarged, thick, firm red-streaked area on the right lobe of the liver of a control rat. The most prevalent finding, common to both control and test item-exposed rats and seen at all examination intervals, was pulmonary in origin and consisted of consolidated areas, small watery-like cysts and shine pinpoint spots on various lobes of the lung.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Chronic respiratory disease was the most prevalent finding and was seen with equal frequency and severity in both control and test item-exposed animals. The presence of hepatic microgranulomata was also noted in control rats and rats exposed to the test substance. All lesions noted microscopically were considered the result of intercurrent disease processes and not as the result of exposure to the test substance.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEC

Effect level:

100 ppm

Based on:

test mat.

Remarks:

= 414 mg/m³

Sex:

male/female

Basis for effect level:

other: Elevations in atypical lymphocyte differentials at day 14 in female rats exposed to 1000 ppm test substance for 7 hours a day

Critical effects observed:

not specified

Lymphocytes

To further clarify the increase in atypical lymphocytes, a recount was performed in which 500 white blood cells were counted and classified by 2 independent observers. Given the agreement between the independent 500 cell recounts, it seems clear that the level of plasmoid lymphocytes was high on day 14 for the 1000 ppm test item 7 hour exposed females. This is in contrast to the less quantitative, original 100 cell differential results which indicated elevations at day 14 for both male and female rats from 100 and 1000 ppm test item groups and at day 28 in the 1000 ppm test item males. In humans, the plasmoid lymphocyte or Turk cell is clinically significant in individuals with viral, parasitic, mycotic or bacterial infections as well as hypersensitive, drug or chemical toxic states. Based upon the information at hand, it is difficult to make a conclusion with respect to the role of test item in producing the changes seen in rats from the present study.

 

Dose levels: conversion from ppm to mg/m³

The following formula was used to convert the dose level in ppm to mg/m³:

dose in mg/m³ = [(gram molecular weight of substance) x (dose in ppm)]/24.45

(reference on site at the sponsor)

Conclusions:

The results of the current study suggest that gross signs of irritation, transient cloudiness of the eye, and possibly elevations in atypical lymphocyte differential counts were the only detectable manifestations of exposure of rats to test substance vapors for five days per week for two weeks.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

414 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral

No reliable repeated dose toxicity data with the test substance is available. Data generated with the related source substance is used for endpoint coverage. A subacute oral study (28 days) is performed with N-ethylmorpholine (NEM), in which male and female rats are exposed to 0 (vehicle), 50, 200, or 800 mg/kg bw/d via gavage (OECD 407). The NOAEL for oral repeated dose toxicity in Cjr:CD(SD)IGS rats is considered to be 50 mg/kg bw/day for both sexes based on cage licking and chewing at 200 and 800 mg/kg bw/d. These clinical signs do not represent a toxicologically adverse effect and are furthermore not mentioned in an OECD 421 study, performed in the same lab with the same rat strain. As relevant adverse effects are observed at 800 mg/kg bw/d only, a NOAEL of 200 mg/kg bw/d is proposed. The justification for read-across within this endpoint can be found in section 13.

A testing proposal for a repeated dose 90-day oral toxicity study in rats is included for the test substance. 

 

Repeated dose toxicity: inhalation

DiPasquale (1979) investigated inhalation toxicity of NMM after repeated whole body exposure for 14 days (1 or 7 hours per day, 5 days per week). Sprague Dawley male and female rats were exposed to 0 (control), 100 or 1000 ppm (eq. 0, 414, 4140 mg/m³; 15 males and 15 females/dose). The results of the current study suggest that gross signs of irritation, transient cloudiness of the eye, and possible elevations in atypical lymphocyte differential counts were the only detectable manifestations of exposure of rats to NMM-vapors for five days per week for two weeks. A NOAEC of 414 mg/m³ was derived. 

 

Repeated dose toxicity: dermal

According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2 adaptation, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the dermal route of exposure.

Justification for classification or non-classification

According to the criteria of the CLP Regulation and based on the study results, the substance should not be classified for STOT repeated exposure.