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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct. 1987 - Jan. 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: NTP-Study: Test procedure in accordance with national standards (NTP)

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988
Reference Type:
other company data
Title:
Unnamed
Year:
1992
Report Date:
1992
Reference Type:
publication
Title:
Toxicity of diethanolamine. 1. Drinking water and topical application exposures in F344 rats
Author:
Melnick RL, et al.
Year:
1994
Bibliographic source:
J. Appl. Toxicol., 14, 1-9
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
no recovery period
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diethanolamine
- Physical state: liquid, colorless to pale yellowish
- Analytical purity: >99% (GC)
- Lot/batch No.: Lot No. A16, Batch No. 02
- Storage condition of test material: room temperature, protected from light
- Source: Kodak Laboratory and Specialty Chemicals (Rochester, NY, USA)

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Taconic Farms, Germantown, NY
- Age at study initiation: 7 weeks
- Fasting period before study: none
- Housing: 5 per cage in polycarbonate cages
- Diet: Zeigler NIH07 Open Formula Diet (Zeigler Brothers, Inc ., Gardners, PA) ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 50 +/- 15
- Air changes (per hr): >12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the drinking water studies, diethanolamine doses were prepared with deionized water as the delivery vehicle; the pH was adjusted to 7.4 ± 0.2 with 1 N hydrochloric acid. Dose solutions were stored no longer than 20 days at room temperature in polypropylene carboys
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The solutions were analyzed by gas chromatography before and after administration to animals.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
320, 630, 1250, 2500, 5000 ppm (males); 160, 320, 630, 1250, 2500 ppm (females)
Basis:
nominal in water
No. of animals per sex per dose:
10 males, 10 females
Control animals:
yes, concurrent no treatment
Details on study design:
Post-exposure period: none
Positive control:
not done

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: twice weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at study termination
- Dose groups that were examined: all dosing groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals

URINALYSIS: Yes
- Time schedule for collection of urine:once in the 12th week
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Sperm morphology and vaginal cytology evaluations
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
Vaginal cytology and sperm morphology evaluations were performed on rats exposed to 0, 63, 125, and 250 mg/kg DEA. Briefly, for the 7 days prior to sacrifice, females were subjected to vaginal lavage with saline. The aspirated cells were scored for the relative preponderance of leukocytes, nucleated epithelial cells, and large squamous epithelial cells to identify the stages of the estrual cycle.

Sperm motility was evaluated at necropsy as follows: Sperm that were extruded from a small cut made in the epididymis were dispersed in a warm, buffered solution, and the number of moving and non-moving sperm in 5 fields of 30 sperm or less per field were counted. After sperm sampling for motility evaluation, the cauda was placed in phosphate buffered saline and incised with a razor blade, the solution mixed gently, then heat-fixed at 65°C. Sperm density was subsequently determined using a hemocytometer. To quantify spermatogenesis, testicular spermatid head count was determined by removing the tunica albuginea and homogenizing the left testis in PBS containing 10% DMSO. Homogenizationresistant spermatid nuclei were enumerated using a hemocytometer.
Statistics:
All numerical data were reported as group means and standard deviations. Body weights, organ weights, and clinical pathology data were tested for homogeneity of variance by Bartlett's test. If the data were non-homogeneous, a separate variance t-test was performed. If the data were homogeneous, a one-way analysis of variance (ANOVA) was performed, followed by Dunnett's test (pairwise comparisons with control).

Analysis of Continuous Variables:
Two approaches were employed to assess the significance of pairwise comparisons between dosed and control groups in the analysis of continuous variables. Organ and body weight data, which are approximately normally distributed, were analyzed using the parametric multiple comparisons procedures of Williams (1971; 1972) and Dunnett (1955). Clinical chemistry and hematology data, which typically have skewed distributions, were analyzed using the nonparametric multiple comparisons methods of Shirley (1977) and Dunn (1964). Jonckheere's test (Jonckheere, 1954) was used to assess the significance of dose-response trends and to determine whether a trend-sensitive test (Williams, Shirley) was more appropriate for pairwise comparisons than a test capable of detecting departures from monotonic dose-response (Dunnett, Dunn). If the P-value from Jonckheere's test was greater than or equal to 0.10, Dunn's or Dunnett's test was used rather than Shirley's or Williams' test.

Results and discussion

Results of examinations

Details on results:
Results summary
Deaths occurred in 2/10 male rats in the top dose group. Surviving animals in the higher concentration groups exhibited depressed weight gains. The animals developed a microcytic anemia in a dose-dependent manner from the lowest dose level onwards. They showed also an increased incidence or severity of nephropathy, tubular necrosis, and mineralization.
Degeneration of the seminiferous tubules of the testis was noted in dosed males and sperm motility and count were decreased at and above 2500 ppm.
Demyelination in the brain and spinal cord was observed in male and female rats at the two highest dose level (2500and 5000 ppm in males; 1250 and 2500 ppm in females).
Finally, the most sensitive parameter was the red blood count and a No Observed Adverse Effect Level (NOAEL) was not achieved for this effect. Consequently, the LOAEL was 320 ppm in males or 160 ppm in females.

Detailed results
Two males in the high dose (5000 ppm) group died before the end of the study. One female death in the lowest dose group (160 ppm) was not considered treatment-related. Body weight gains were depressed in a dose-related fashion in both sexes. Decreased water consumption among the higher dose groups may have contributed in part to the decreased body weight gain. Based on water consumption and body weight data, average daily doses of diethanolamine were estimated to range from about 25 to 440 mg/kg in males and about 15 to 240 mg/kg in females.

Clinical signs of toxicity included tremors, emaciation, abnormal posture, and rough hair coat in the 2 highest dose groups of each sex. Diethanolamine administration produced a moderate, poorly regenerative, microcytic, normochromic anemia in male and female rats. Hematologic effects were dose-dependent and included decreases in erythrocyte and reticulocyte counts, hemoglobin concentration, hematocrit, MCV, and MCH. MCV was reduced in rats at all dose levels. Hematologic effects were not associated with microscopic changes in the femoral bone marrow. No significant gross lesions attributable to diethanolamine were found at necropsy.

Dose-related increases in relative kidney weights were observed in males and females. Kidney weight changes were accompanied by increases in the incidence and/or severity of nephropathy, renal tubular cell necrosis, or tubular mineralization. Nephropathy consisted of tubules lined by epithelial cells with more basophilic staining of the cytoplasm and a higher nuclear/cytoplasmic ratio; occasionally, thickened basement membranes were seen around these tubules. This lesion was present to a minimal degree in controls, particularly in male rats, but was increased in incidence and severity in high dose males and in most female treatment groups. Increased nephropathy was considered a regenerative change and was supported by the observation of tubular necrosis at the higher doses. Tubular necrosis was minimal in severity and was characterized by eosinophilic tubular epithelial cells with pyknotic nuclei, frequently seen desquamated into the lumen of renal tubules. Mineralization was observed as basophilic concretions within necrotic tubules which were present primarily along the outer stripe of the outer medulla. Mineralization was present in all female control rats; however, there was a dose-related increase in severity and/or incidence in both females and males.

The brain and spinal cord also were identified as targets of diethanolamine toxicity. In the brain, microscopic change was observed in coronal sections of the medulla oblongata and consisted of bilaterally symmetrical areas of vacuolization of the neuropil. Vacuoles were most consistently seen as sharply delimited, round-to-oval, clear spaces arranged symmetrically around the midline of the medulla in areas of transversely sectioned white matter identified as the tectospinal tract. In more severe cases, there was involvement of more peripheral white matter tracts at the same level of the medulla. Generally, vacuoles were empty and not associated with a glial response, although some contained debris, and a minimal cellular reaction was present. Special stains for myelin demonstrated only a focal loss of myelin sheaths in these vacuolated areas. In transverse sections of the spinal cord, vacuoles were randomly scattered in the dorsal, ventral, and lateral columns of the white matter and in spinal nerves. No lesions were observed in sections of the sciatic nerve. Minimal to mild demyelination of the brain and spinal cord was observed in all male and female rats in the 2500 and 5000 ppm dose groups. There were no neurologic clinical signs that could be clearly attributed to these lesions.

Decreases in testis and epididymis weights were associated microscopically with degeneration of seminiferous epithelium and with hypospermia. The testicular lesion was morphologically similar to that seen in the 2-week drinking water study and consisted of decreased numbers of spermatogenic cells, reduced size of seminiferous tubules, and scant intraluminal sperm. Testicular degeneration was diagnosed in all high dose (5000 ppm) males and in 3 of 10 males at the 2500 ppm dose level. Intraluminal cellular debris and reduced numbers of sperm cells were present in the epididymis. These findings correlated with decreases in sperm motility and sperm count per gram caudal tissue. Atrophy of the seminal vesicles and prostate glands in male rats from the higher dose groups were additional treatment-related lesions. There were no noteworthy changes among female rats in estrous cycle length.

Cytoplasmic vacuolization of the zona glomerulosa of the adrenal cortex was a treatment-related effect in high dose male rats (9 of 10) and in females in the 2500 (2 of 10) and 5000 ppm (10/10) dose groups. This was a minimal change consisting of small clear vacuoles in the cytoplasm of these cells and may have been related to increased mineralocorticoid production secondary to renal damage and/or dehydration.

Dose-related increases in relative liver weights occurred in male and female rats. Although the changes in liver weights were not associated with microscopic lesions in the liver, there were mild to moderate increases in serum concentrations of total bile acids in female rats in all dose groups, and in male rats in all dose groups except the lowest (320 ppm).

Other relevant biochemical changes in male and female rats included increases in concentrations of albumin, total protein, and UN in serum. Treatment-related microscopic lesions in the 2 high-dose group male rats that died before study termination were similar to those of rats that survived to the end of the study.

Effect levels

open allclose all
Dose descriptor:
LOAEL
Effect level:
160 ppm
Sex:
female
Basis for effect level:
other: haematology, nephrotoxicity (f), kidney weight↑
Dose descriptor:
LOAEL
Effect level:
14 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: haematology, nephrotoxicity (f), kidney weight↑
Dose descriptor:
LOAEL
Effect level:
320 ppm
Sex:
male
Basis for effect level:
other: haematology, kidney weight↑
Dose descriptor:
LOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: haematology, kidney weight↑

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Survival, weight gain and water consumption

Dose (ppm)

Survivala

Weight gainb

Final weight relative to controlsc

Average water consuptiond

Estimated DEA consumede

MALE

0

10/10

240

-

20.9

0

320

10/10

221

95

20.2

25

630

10/10

200

89

19.2

48

1250

10/10

180

82

18.3

97

2500

10/10

135

71

17.7

202

5000

8/10

81

56

15.6

436

FEMALE

0

10/10

120

-

15.5

0

160

9/10

106

95

14.9

14

320

10/10

98

91

16.9

32

630

10/10

95

90

15.2

57

1250

10/10

85

84

15.8

124

2500

10/10

63

75

13.9

242

a Number surviving at 13 weeks/number of animals per dose group.

b Mean weight change of the animals in each dose group.

c (Dosed group mean/Control group mean) x 100.

d Average water consumption in ml/animal/day.

e Chemical consumption in mg/kg body weight/day.

Hematological changes in peripheral blood

Dose (ppm)

0

160a

320

630

1250

2500

5000b

MALE

RBC (106/ml)

8.79

-

8.75

8.20**

7.33**

6.40**

5.71**

HGB (g/dl)

14.8

-

14.3*

13.3**

11.6**

9.8**

8.9**

HCT (%)

47.8

-

46.1

42.5**

36.9**

31.4**

27.8**

MCV (fl)

54

-

53**

52**

50**

49**

49**

MCH (pg)

16.9

-

16.4**

16.2**

15.9**

15.3**

15.5**

Reticulocytes (106/µl)

0.23

-

0.23

0.23

0.24

0.14**

0.16**

FEMALE

RBC (106/ml)

8.40

8.51

7.84**

7.56**

6.78**

6.43**

-

HGB (g/dl)

15.1

15.2

13.8**

13.0**

11.3**

10.5**

-

HCT (%)

47.3

47.0

42.3**

39.7**

34.4**

31.2**

-

MCV (fl)

56

55**

54**

53**

51**

49**

-

MCH (pg)

17.9

17.8*

17.7**

17.2**

16.7**

16.3**

-

Reticulocytes (106/µl)

0.17

0.16

0.13**

0.12*

0.09**

0.08**

-

a Females only, N = 9.

b Males only, N = 8.

* Significantly different from control (p ≤ 0.05) by Dunn's or Shirley's test.

** Significantly different from control (p ≤ 0.01) by Dunn's or Shirley's test.

Organ weights

Dose (ppm)

0

160

320

630

1250

2500

5000

MALE

Necropsy body weight

366

-

339

326

302

265

205

Kidney weight

1.29

-

1.34

1.30

1.21

1.18

1.26

Relative kidney weight

3.54

-

3.94**

3.99**

3.98**

4.44**

6.14**

Liver weight

15.09

-

13.87

14.92

14.82**

14.18

11.59**

Relative liver weight

41.28

-

40.79

45.61**

48.90

53.27**

56.71**

Right testis weight

1.49

-

1.46

1.47

1.27**

0.97**

0.54**

Relative testis weight

4.08

-

4.31

4.50

4.22

3.64**

2.63**

Epididymis weight

0.426

-

0.453

0.392

0.309**

0.184**

0.134**

Relative epididymis weight

1.17

-

1.34**

1.20

1.02**

0.68.**

0.65**

FEMALE

Necropsy body weight

218

208

201

202

188

162

-

Kidney weight

0.66

0.86**

0.84**

0.83*

0.87**

0.92**

-

Relative kidney weight

3.03

4.12

4.21**

4.12**

4.63**

5.67**

-

Liver weight

6.08

6.36

7.04**

6.99**

7.78**

7.32**

-

Relative liver weight

27.86

30.54

35.09**

34.52**

41.41**

45.26**

-

a Organ weights and body weights given in grams; organ-weight-to-body-weight ratios given as mg organ weight/g body weight.

* Significantly different from the control group by Williams' or Dunnett's test (P≤0.05).

** Significantly different from the control group by Williams' or Dunnett's test (P≤0.01).

Incidence and severity of kidney, brain and spinal cord lesions

Dose (ppm)

0

160

320

630

1250

2500

5000

MALE

Kidney

Nephropathy

6/10 (1.0)

-

2/10 (1.0)

2/10 (1.0)

3/10 (1.0)

6/10 (1.0)

10/10 (2.4)

Tubular epithelial necrosis

0/10

-

0/10

0/10

0/10

0/10

10/10 (1.0)

Tubular mineralization

0/10

-

0/10

0/10

1/10 (1.0)

10/10 (1.8)

10/10 (1.7)

Brain, medulla

Demyelination

0/10

-

0/10

0/10

0/10

10/10 (1.7)

10/10 (2.0)

Spinal cord

Demyelination

0/10

-

0/10

0/10

0/10

10/10 (1.9)

10/10 (2.0)

FEMALE

Kidney

Nephropathy

2/10 (1.0)

9/10 (1.0)

10/10 (1.5)

10/10 (1.4)

9/10 (1.0)

2/10 (1.0)

-

Tubular epithelial necrosis

0/10

0/10

0/10

0/10

1/10 (1.0)

3/10 (1.0)

-

Tubular mineralization

10/10 (1.3)

10/10 (2.0)

10/10 (2.5)

10/10 (3.0)

10/10 (2.4)

10/10 (1.7)

-

Brain, medulla

Demyelination

0/10

0/10

0/10

0/10

10/10(1.5)

10/10(1.9)

-

Spinal cord

Demyelination

0/10

0/10

0/10

0/10

10/10(1.0)

10/10(1.9)

-

Applicant's summary and conclusion