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Repeated dose toxicity: dermal

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Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: NTP-study according to national standards

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988
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:
1994
Reference Type:
publication
Title:
Unnamed
Year:
1992
Report Date:
1992
Reference Type:
other company data
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
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:
TEST ANIMALS
- Source: Taconic Farms, Germantown, NY
- Age at study initiation: 7 weeks
- Weight at study initiation: mean weight males: 94.6g; mean weight females: 88.8g
- Fasting period before study: none
- Housing: indivilually in polycarbonate cages
- Diet: Zeigler NIH07 Open Formula Diet (Zeigler Brothers, Inc ., Gardners, PA) ad libitum
- Water: ad libitum
- Acclimation period: 13 days


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


Administration / exposure

Type of coverage:
open
Vehicle:
ethanol
Details on exposure:
Route of Administration: dermal
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing formulations were analysed by gas chromatography before and after administration to animals.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
once daily, 5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 32, 63, 125, 250, 500 mg/kg bw
Basis:
nominal per unit body weight
No. of animals per sex per dose:
10 males, 10 females
Control animals:
yes, concurrent vehicle
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:
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 1-way analysis of variance (ANOVA) was performed, followed by Dunnett's test (pairwise comparisons with control)
Organ and body weight data were analyzed using the parametric multiple comparisons procedures of Williams and Dunnett.  Clinical chemistry data 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. The outlier test of Dixon and Massey (1951) was employed to detect extreme values. 
Analysis of Vaginal Cytology data 
Since the data are proportions, an arcsine transformation was used to bring the data into closer conformance withnormality assumptions. Treatment effects were investigated by applying a multivariate analysis of variance to the transformed data to test for the simultaneous equality of measurements across dose levels.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
One male animal of the 500 mg/kg dosing group died during week 9, and 2 females administered 500 mg/kg/day diethanolamine were killed in a moribund condition during week 10.

The primary clinical signs of toxicity in the 3 highest dosing groups were irritation and crusting of the skin at the site of diethanolamine application.

BODY WEIGHT AND WEIGHT GAIN
Final mean body weights of males receiving doses of 250 or 500 mg/kg, and of females receiving doses of 125 mg/kg or higher, were lower than those of controls.

HAEMATOLOGY
A moderate microcytic, normochromic anemia developed in male and female rats. Decreases in red blood cell variables were observed even at the lowest dose, 32 mg/kg. No histological changes in femoral bone marrow  were observed.

CLINICAL CHEMISTRY
Serum biochemical changes in male rats included mild increases in concentrations of UN and albumin at the 4th and 2nd highest dose groups, respectively, and mild increases in activities of ALT in animals in the 3 highest dose groups.
In female rats, UN, albumin, and total protein increased in all dose groups (except at the lowest dose for total protein), and total bile acids increased in the 2 highest dose groups. A mild increase in activity of ALT occurred in female rats in the highest dose group.

ORGAN WEIGHTS
Effects were noted for the kidneys in form of an increase in absolute and relative kidney weights in male and female rats. These weight changes  were associated with increased severity or increased incidences of nephropathy, renal tubular cell necrosis, or tubular mineralization. A dose-dependent increase in incidence and severity of nephropathy was evident at the lower dose levels in females, but there was no clear treatment effect on this lesion in male rats.
There was a dose-dependent increase in absolute and relative liver weights in both male and female rats associated with mild changes in clinical chemistry but no corresponding microscopic lesion was observed. 
Sperm morphology and vaginal cytology evaluations did not show adverse  effects.

GROSS PATHOLOGY
nothing abnormal detected except skin lesions described below

HISTOPATHOLOGY: NON-NEOPLASTIC
Demyelination in the medulla oblongata was observed in all males and  females in the 500 mg/kg dose group, and in 7 females in the 250 mg/kg dose group, however, all lesions were minimal in severity and there was no spinal cord involvement.

SKIN LESIONS:
Skin lesions in form of ulceration were noted and ranged from small, superficial foci of epidermal loss to extensive areas of coagulation necrosis of the epidermis and dermis. The ulcers were accompanied by inflammatory cell infiltration that was prominent at the borders between necrotic and viable tissue. Minimal to moderate acanthosis (epidermal hyperplasia) invariably was present at ulcer margins in the higher dose groups; at lower dose levels, only minimal acanthosis and  hyperkeratosis were present.

OTHER FINDINGS:
All early-death rats in this study had lesions of the kidney, skin, and brain as described above. The severity of these lesions, however, was no greater than was seen in animals that survived to the end of the study.

Dermal exposure to diethanolamine was not associated with testicular or epididymal changes. Sperm morphology and vaginal cytology evaluations did not show adverse effects.

Effect levels

Dose descriptor:
LOAEL
Effect level:
32 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: haematological changes, nephropathy and hyperkeratosis of the skin

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

One 500 mg/kg male died during week 9, and 2 females administered 500 mg/kg diethanolamine were killed in a moribund condition during week 10.

Final mean body weights of males receiving doses of 250 or 500 mg/kg, and of females receiving doses of 125 mg/kg or higher, were lower than those of controls.

The primary clinical signs of toxicity in the 3 highest dose groups were irritation and crusting of the skin at the site of diethanolamine application. A moderate, poorly regenerative, microcytic, normochromic anemia developed in male and female rats exposed dermally to diethanolamine for 13 weeks. Decreases in red blood cell variables were observed even at the lowest dose, 32 mg/kg; thus, a no-observable-adverse-effect level (NOAEL) for diethanolamine-induced anemia was not achieved. No histologic changes in femoral bone marrow were observed.

Serum biochemical changes in male rats included mild increases in concentrations of UN and albumin at the 4th and 2nd highest dose groups, respectively, and mild increases in activities of ALT in animals in the 3 highest dose groups. In female rats, UN, albumin, and total protein increased in all dose groups (except at the lowest dose for total protein), and total bile acids increased in the 2 highest dose groups. A mild increase in activity of ALT occurred in female rats in the highest dose group.

The kidney was identified as a target organ in the 13-week dermal study. Absolute and relative kidney weights were increased in male and female rats. These weight changes were associated with increased severity or increased incidences of nephropathy, renal tubular cell necrosis, or tubular mineralization. A dose-dependent increase in incidence and severity of nephropathy was evident at the lower dose levels in females, but there was no clear treatment effect on this lesion in male rats as there was in the 13-week drinking water study. Tubular necrosis was observed in females in the 2 highest dose groups, but no active necrosis was found in the corresponding male groups. Tubular mineralization, consistent with previous necrosis, was present in high-dose males, as well as being increased in incidence and severity in most treated female groups.

There was a dose-dependent increase in absolute and relative liver weights in both male and female rats. Although mild serum biochemical changes occurred, no corresponding microscopic lesion was observed.

Dermal exposure to diethanolamine was not associated with testicular or epididymal changes. Sperm morphology and vaginal cytology evaluations did not show adverse effects.

Lesions of the treated skin were similar to those present in the 2-week study, and were dose related in incidence and severity. The lesion was diagnosed as ulceration and ranged from small, superficial foci of epidermal loss to extensive areas of coagulation necrosis of the epidermis and dermis. The ulcers were accompanied by inflammatory cell infiltration that was prominent at the borders between necrotic and viable tissue. Inflammation was primarily neutrophilic, but was designated "chronic-active" due to the frequent appearance of fibrovasculartissue proliferation in the vicinity of ulcers. Minimal to moderate acanthosis (epidermal hyperplasia) invariably was present at ulcer margins in the higher dose groups; at lower dose levels, only minimal acanthosis and hyperkeratosis were present. Demyelination in the medulla oblongata was observed in all males and females in the 500 mg/kg dose group, and in 7 females in the 250 mg/kg dose group. The lesion was morphologically and topographically similar to that diagnosed in the 13-week drinking water study; it was characterized by intramyelinic vacuoles arranged symmetrically around the medial medulla oblongata in the region of the tectospinal tract. Unlike the drinking water study, however, all lesions were minimal in severity and there was no spinal cord involvement. All early-death rats in this study had lesions of the kidney, skin, and brain as described above. The severity of these lesions, however, was no greater than was seen in animals that survived to the end of the study.

Mean body weight gains of F344/N rats in the 13-week dermal studies of diethanolamine

Dose

(mg/kg bw/day)

mean body weight gain (g)

final weight relative to controls (%)

males

0

218

-

32

215

99

63

200

94

125

216

98

250

170**

86

500

117**

69

females

0

85

-

32

80

100

63

77

99

125

72**

93

250

63**

90

500

42**

79

** p<0.01

Haematological changes in peripheral blood of F344/N rats in the 13-week dermal studies of diethanolamine

Dose (mg/kg)

0

32

63

125

250

500

Males

RBC (106/μL)

8.87

8.81

8.79

8.57*

7.90**

6.80**

HGB (g/dL)

15.5

15.3

15.1*

14.3**

12.9**

11.0**

HCT (%)

47.6

46.4

45.6*

43.1**

38.8**

32.6**

MCV (fL)

54

53**

52**

50**

49**

48**

MCH (pg)

17.5

17.3**

17.1**

16.7**

16.3**

16.1**

Reticulocytes (106/μL)

0.20

0.21

0.20

0.21

0.18

0.18

Females

RBC (106/μL)

8.45

8.14**

7.83**

7.38**

6.91**

6.23**

HGB (g/dL)

15.5

14.8**

14.1**

13.2**

12.0**

10.5**

HCT (%)

48.9

46.7**

44.2**

40.6**

36.9**

31.9**

MCV (fL)

58

57

56**

55**

53**

51**

MCH (pg)

18.4

18.2

18.1*

17.8**

17.4**

16.8**

Reticulocytes (106/μL)

0.16

0.13*

0.12**

0.10**

0.14*

0.12**

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

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


Kidney and liver weights of F344/N rats administered diethanolamine dermally for 13 weeks

Dose (mg/kg)

0

32

63

125

250

500

Males

relative kidney weight

3.39**

4.12

3.68**

3.87**

4.04**

5.38**

relative liver weight

38.2

41.2*

40.5**

46.6**

50.3**

58.3**

Females

relative kidney weight

3.59

5.00**

4.69**

5.12**

5.25**

6.83**

relative liver weight

33.5

38.9**

39.7**

43.4**

47.1**

58.4**

Relative organ weights given in mg organ weight/gm body weight

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

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

Dose (mg/kg)

0

32

63

125

250

500

Males

Kidney

Nephropathy

9/10 (1.0)

6/10 (1.0)

5/10 (1.0)

6/10 (1.0)

4/10 (1.0)

5/10 (1.0)

Tubular epithelial necrosis

0/10

0/10

0/10

0/10

0/10

0/10

Tubular mineralization

0/10

0/10

0/10

0/10

0/10

9/10 (1.9)

Brain, Medulla

Demyelination

0/10

0/10

0/10

0/10

0/10

10/10 (1.0)

Skin

Ulcer

0/10

0/10

0/10

0/10

3/10 (1.3)

10/10 (2.6)

Chronic active inflammation

0/10

0/10

0/10

0/10

3/10 (1.3)

10/10 (1.7)

Acanthosis

0/10

0/10

3/10 (1.0)

6/10 (1.0)

6/10 (1.5)

10/10 (2.2)

Hyperkeratosis

0/10

0/10

5/10 (1.0)

10/10 (1.1)

10/10 (1.4)

10/10 (1.9)

Females

Kidney

Nephropathy

3/10 (1.0)

9/10 (1.3)

10/10 (1.4)

10/10 (1.7)

7/10 (1.1)

4/10 (1.0)

Tubular epithelial necrosis

0/10

0/10

0/10

0/10

2/10 (1.0)

10/10 (1.0)

Tubular mineralization

4/10 (1.0)

9/10 (1.0)

10/10 (1.6)

10/10 (1.9)

10/10 (1.1)

10/10 (1.0)

Brain, Medulla

Demyelination

0/10

0/10

0/10

0/10

7/10 (1.0)

9/10 (1.0)

Skin

Ulcer

0/10

0/10

0/10

1/10 (1.0)

7/10 (1.9)

10/10 (3.4)

Chronic active inflammation

0/10

0/10

0/10

3/10 (1.0)

7/10 (1.6)

10/10 (2.5)

Acanthosis

0/10

0/10

1/10 (1.0)

6/10 (1.2)

7/10 (2.0)

10/10 (2.6)

Hyperkeratosis

0/10

5/10 (1.0)

6/10 (1.0)

9/10 (1.2)

10/10 (1.7)

10/10 (2.1)

Incidence and severity score ( ) based on a scale of 1 to 4: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. Severity scores are averages

based on the number of animals with lesions.

Applicant's summary and conclusion