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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
October 1986 to December 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results. Reliability of 2 given since the data is based on read across, not the target substance.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1992
Reference Type:
publication
Title:
Toxicity of Diethanolamine. 1. Drinking Water and Topical Application Exposures in F344 Rats
Author:
Melnick, R.L., Mahler, J., Bucher, J.R., Thompson, M., Hejtmancik, M., Ryan, M.J. & Mezza, L.E.
Year:
1994
Bibliographic source:
J. Appl. Toxicol. 14, 1-9 (1994)

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: ca. 7 weeks of age
- Weight at study initiation: Males had a mean initial bodyweight of 120 to 124 g; females had a mean initial bodyweight of 106 to 114 g.
- Housing: animals were housed individually.
- Diet (e.g. ad libitum): diet in pellet form was available ad libitum.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 20 to 24 °C (72 ± 3 °F)
- Humidity (%): 50 ± 15 % relative humidity
- Air changes (per hr): 10 to 12 fresh-air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h per day of subdued fluorescent light

Administration / exposure

Type of coverage:
open
Vehicle:
ethanol
Details on exposure:
TEST SITE
- Area of exposure: The dosing solution was applied to the shaved back of each animal from the mid-back to the interscapular region using a calibrated micropipette.
- Time intervals for shavings or clippings: Rats were shaved at 1 week intervals.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The volume of the dosing solution was adjusted weekly based on the most recent mean body weight of each dose group at concentrations of 0, 37.5, 75, 150, 300 and 600 mg/mL (0, 32, 63, 125, 250 and 500 mg/kg)
- Concentration (if solution): The test material was administered as a 95 % solution in ethanol.

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were analysed by gas chromatography before and after administration to animals and found to be within 10 % of the theoretical values.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once per day, except for weekends and holidays
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 32, 63, 125, 250 and 500 mg/kg
Basis:
nominal per unit body weight
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected on the basis of a two-week study conducted prior to the 13 week study.
In the two week study, rats of both sexes received the test material in ethanol (95 %) at doses of 0, 63, 125, 250, 500 and 1000 mg/mL with target doses of 0, 125, 250, 500, 1000, and 2000 mg/kg bodyweight. Early deaths of male rats occurred in the highest dose group and in female rats in the 2 highest dose groups (1000 and 2000 mg/kg). Bodyweight gains were reduced in the higher dose groups.
Animals exhibited ulcerative skin lesions at the site of application, accompanied by inflammatory cell infiltration, hyperkeratosis, and acanthosis (hyperplasia) of the epidermis. Hyperkeratosis, without ulceration, was observed in some animals.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule for examinations: Animals were examined twice daily for mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were recorded weekly and at necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Observations were recorded weekly and at necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: No

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was measured twice weekly.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the study, blood samples were collected in Microtainers containing dipotassium EDTA and analysed with an Ortho ELT-8 laser haematology counter (Ortho Instruments, Westwood, MA).
- Anaesthetic used for blood collection: Yes. Animals were anesthetized with carbon dioxide and blood samples were collected from the retroorbital sinus.
- Animals fasted: No data
- How many animals: Clinical pathology studies were performed on all rats that survived until the end of the study.
- Parameters evaluated included: erythrocyte count (RBC), leukocyte count (WBC), mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), haemoglobin (HGB), haematocrit (HCT), differential leukocyte count, erythrocyte morphological assessment, reticulocyte count, platelet count and platelet morphological assessment.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the study, biochemical analyses were performed on blood samples collected in Microtainers (Becton Dickinson, Rutherford, NJ) with no preservative or anticoagulant And analysed using a Hitachi 704 automatic chemistry analyser (Boehringer-Mannheim Diagnostics, Indianapolis, IN).
- Anaesthetic used for blood collection: Yes. Animals were anesthetized with carbon dioxide and blood samples were collected from the retroorbital sinus.
- Animals fasted: No data
- How many animals: Clinical pathology studies were performed on all rats that survived until the end of the study.
- Parameters evaluated included: serum sorbitol dehydrogenase (SDH), alanine arninotransferase (ALT), total protein (TP), albumin, urea nitrogen (UN), creatinine, glucose and total bile acids.

URINALYSIS: Yes
- Time schedule for collection of urine: During the 12th week of the study.
- Metabolism cages used for collection of urine: Yes. Urine samples were collected over a 16 hour period from rats housed individually in polycarbonate metabolism cages.
- Animals fasted: Yes, food was removed from the cages.
- Methods: Collection tubes were immersed in ice-water baths.
- Parameters evaluated: Volume, appearance, specific gravity and pH were measured for each urine sample. Concentrations of glucose, protein, urea nitrogen and creatinine, and activities of alkaline phosphatase and lactate dehydrogenase, were measured using a Hitachi 704 chemistry analyser (Boehringer-Mannheim Diagnostics, Indianapolis, IN).

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Complete necropsies were performed on all animals. The brain, heart, right kidney, liver, lung, right testis and thymus were weighed. Organs and tissues were examined for gross lesions and fixed in 10 % neutral buffered formalin. Tissues were trimmed, embedded in paraffin, sectioned, stained with haematoxylin and eosin and examined microscopically.

HISTOPATHOLOGY: Yes. Complete histopathological examinations were performed on all control animals, all early death animals and all animals in the highest dose groups with at least 60 % survivors. Target tissues were examined in animals from lower dose groups until a no-effect level was determined. All lesions observed at necropsy were examined microscopically.
These tissues included: adrenal glands, brain (3 sections), clitoral glands, eyes (if grossly abnormal), bone (femur, sternebrae, or vertebrae) with marrow, gross lesions, heart/aorta, intestine-large (cecum, colon, rectum), intestine-small (duodenum, jejunum, ileum), kidneys, liver, lung/mainstem bronchi, lymph nodes (mandibular, mesenteric), mammary gland, nasal cavity and turbinates (3 sections), oesophagus, ovaries, pancreas, parathyroid glands, pituitary gland, preputial glands, prostate gland, salivary glands, seminal vesicles, skin (skin sections of gross lesions at the site of application, skin at the site of application without gross lesions, and undosed inguinal control skin), spinal cord and sciatic nerve, spleen, stomach (forestomach and glandular stomach), testes with epididymis, thymus, thyroid gland, trachea, urinary bladder, and uterus.
Other examinations:
Vaginal cytology and sperm morphology evaluations were performed, using the methods described by Morrissey et al. (1988) on animals receiving 0, 63, 125 and 250 mg/kg.
- 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: 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 homogenising the left testis in PBS containing 10 % DMSO. Homogenisation-resistant spermatid nuclei were enumerated using a haemocytometer.
Statistics:
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 analysed using the parametric multiple comparisons procedures of Williams (1971; 1972) and Dunnett (1955). Clinical chemistry and haematology data were analysed using the nonparametric multiple comparisons methods of Shirley (1977) and Dunn (1964). Jonckheere's test (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. No value selected by the outlier test was eliminated unless it was at least twice the next largest value or at most half of the next smallest value.

Analysis of Vaginal Cytology data
Since the data are proportions (the proportion of the observation period that an animal was in a given estrous state), an arcsine transformation was used to bring the data into closer conformance with normality assumptions. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for the simultaneous equality of measurements across dose levels.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see below
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
see below
Mortality:
mortality observed, treatment-related
Description (incidence):
see below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below
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):
see below
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see below
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below
Histopathological findings: neoplastic:
no effects observed
Details on results:
One 500 mg/kg male died during week 9 and 2 females administered 500 mg/kg were killed in a moribund condition during week 10 (Table 1). Final mean bodyweights 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 test material application.
A moderate, poorly regenerative, microcytic, normochromic anaemia developed in male and female rats (Table 2). 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 test material-induced anaemia 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: absolute and relative kidney weights were increased in male and female rats (Table 3). These weight changes were associated with increased severity or increased incidences of nephropathy, renal tubular cell necrosis or tubular mineralisation (Table 4). 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 males.
Tubular necrosis was observed in females in the 2 highest dose groups, but no active necrosis was found in the corresponding male groups. Tubular mineralisation, 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 (Table 3). Although mild serum biochemical changes occurred, no corresponding microscopic lesion was observed. Dermal exposure 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 dose-related in incidence and severity (Table 4). 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 fibrovascular tissue 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 (Table 4). The lesion was characterised by intramyelinic vacuoles arranged symmetrically around the medial medulla oblongata in the region of the tectospinal tract. 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.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: A NOAEL was not achieved for the haematological changes.
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
LOAEL
Effect level:
32 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based upon haematological changes, nephropathy and hyperkeratosis of the skin.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1 Summary of Survival and Weight Gain

Sex

Dose

(mg/kg)

Survival

Mean Bodyweight

(g)

Final Weight Relative to Controls (%)

Initial

Final

Change

 

 

Male

0

32

63

125

250

500

10/10

10/10

10/10

10/10

10/10

9/10

124

122

123

120

124

120

342

337

323

336

294

237

218

215

200

216

170

117

-

99

94

98

86

69

 

 

Female

0

32

63

125

250

500

10/10

9/10

10/10

10/10

10/10

8/10

107

113

114

106

109

109

192

193

191

178

172

151

85

80

77

72

63

42

-

100

99

93

90

79

Survival is represented as the number of animals surviving at 13 weeks / number of animals per dose group

 

Table 2 Haematological Changes in Peripheral Blood

Sex

Parameter

Dose (mg/kg)

0

32

63

125

250

500

 

 

Male

RBC (10/µL)

HGB (g/dL)

HCT (%)

MCV (fL)

MCH (pg)

Reticulocytes (10/µL)

8.87

15.5

47.6

54

17.5

0.20

8.81

15.3

46.4

53**

17.3**

0.21

8.79

15.1*

45.6*

52**

17.1**

0.20

8.57*

14.3**

43.1**

50**

16.7**

0.21

7.90**

12.9**

3838**

49**

16.3**

0.18

6.80**

11.0**

32.6**

48**

16.1**

0.18

 

 

Female

RBC (10/µL)

HGB (g/dL)

HCT (%)

MCV (fL)

MCH (pg)

Reticulocytes (10/µL)

8.45

15.5

48.9

58

18.4

0.16

8.14**

14.8**

46.7**

57

18.2

0.13*

7.83**

14.1**

44.2**

56**

18.1*

0.12**

7.38**

13.2**

40.6**

55**

17.8**

0.10**

6.91**

12.0**

36.9**

53**

17.4**

0.14*

6.23**

10.5**

31.9**

51**

16.8**

0.12**

* = Significantly different from control group (p 0.05) by Dunn’s or Shirley’s test

** = Significantly different from control group (p 0.01) by Dunn’s or Shirley’s test

 

Table 3 Kidney and Liver Weights

Organ weights and bodyweights are given in grams; organ weight to bodyweight ratios are given as mg organ weight/g bodyweight

Sex

Parameter

Dose (mg/kg)

0

32

63

125

250

500

 

 

Male

Necropsy weight

Kidney Weight

Relative kidney weight

Liver Weight

Relative liver weight

347

1.17

3.39

13.25

38.2

342

1.41**

4.12**

14.10

41.2*

328

1.21

3.68**

13.29

40.5**

342

1.32*

3.87**

16.00*

46.6**

300

1.20

4.04**

15.12*

50.3**

241**

1.31

5.38**

14.05*

58.3**

 

 

Female

Necropsy weight

Kidney Weight

Relative kidney weight

Liver Weight

Relative liver weight

193

0.69

3.59

6.48

33.5

194

0.97**

5.00**

7.56**

38.9**

191

0.90**

4.69**

7.59**

39.7**

180**

0.92**

5.12**

7.79**

43.4**

174**

0.91**

5.25**

8.17**

47.1**

154**

1.05**

6.83**

9.00**

58.4**

* = Significantly different from control group (p 0.05) by Williams’ or Dunnett’s test

** = Significantly different from control group (p 0.01) by Williams’ or Dunnett’s test

 

Table 4 Incidence and Severity of Kidney, Brain and Skin Lesions

Sex

Parameter

Dose (mg/kg)

0

32

63

125

250

500

 

 

Male

Kidney

Nephropathy

Tubular epithelial necrosis

Tubular mineralisation

Brain, medulla

Demyelination

Skin

Ulcer

Chronic active inflammation

Acanthosis

Hyperkeratosis

 

9/10 (1.0)

0/10

0/10

 

0/10

 

0/10

0/10

0/10

0/10

 

6/10 (1.0)

0/10

0/10

 

0/10

 

0/10

0/10

0/10

0/10

 

5/10 (1.0)

0/10

0/10

 

0/10

 

0/10

0/10

3/10 (1.0)

5/10 (1.0)

 

6/10 (1.0)

0/10

0/10

 

0/10

 

0/10

0/10

6/10 (1.0)

10/10 (1.1)

 

4/10 (1.0)

0/10

0/10

 

0/10

 

3/10 (1.3)

3/10 (1.3)

6/10 (1.5)

10/10 (1.4)

 

5/10 (1.0)

0/10

9/10 (1.9)

 

10/10 (1.0)

 

10/10 (2.6)

10/10 (1.7)

10/10 (2.2)

10/10 (1.9)

 

 

Female

Kidney

Nephropathy

Tubular epithelial necrosis

Tubular mineralisation

Brain, medulla

Demyelination

Skin

Ulcer

Chronic active inflammation

Acanthosis

Hyperkeratosis

 

3/10 (1.0)

0/10

4/10 (1.0)

 

0/10

 

0/10

0/10

0/10

0/10

 

9/10 (1.3)

0/10

9/10 (1.0)

 

0/10

 

0/10

0/10

0/10

5/10 (1.0)

 

10/10 (1.4)

0/10

10/10 (1.6)

 

0/10

 

0/10

0/10

1/10 (1.0)

6/10 (1.0)

 

10/10 (1.7)

0/10

10/10 (1.9)

 

0/10

 

1/10 (1.0)

3/10 (1.0)

6/10 (1.2)

9/10 (1.2)

 

7/10 (1.1)

2/10 (1.0)

10/10 (1.1)

 

7/10 (1.0)

 

7/10 (1.9)

7/10 (1.6)

7/10 (2.0)

10/10 (1.7)

 

4/10 (1.0)

10/10 (1.0)

10/10 (1.0)

 

9/10 (1.0)

 

10/10 (3.4)

10/10 (2.5)

10/10 (2.6)

10/10 (2.1)

The severity score, represented by ( ), is 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

Conclusions:
A No-Observed-Adverse-Effect Level (NOAEL) was not achieved for the haematological changes. On this basis, it is considered that the LOAEL level for rats is 32 mg/kg bw/day and as a result the test material requires classification as STOT RE Category 2 in accordance with EU criteria.
Executive summary:

The repeated dose toxicity of the test material was investigated in a procedure equivalent to the standardised guideline OECD 411 under GLP conditions.

The test material was administered to male and female F344 rats for 13 weeks' duration, 5 days per week, to the shaved interscapular region. Doses ranged from 32 to 500 mg/kg bw/day prepared in 95 % ethanol.

Early deaths were observed in the highest dose groups and bodyweight gains were reduced in animals given the higher doses. Animals exhibited dose-dependent haematologic and renal function changes, in addition to skin lesions at the site of application which included ulceration and inflammation, hyperkeratosis, and acanthosis. Liver weights were increased in males and females, but there were no associated histopathological changes.

Other treatment-related effects observed included demyelination in the brain and spinal cord and nephropathy, renal tubular necrosis, and/or tubular mineralisation.

A No-Observed-Adverse-Effect Level (NOAEL) was not achieved for the haematological changes. On this basis, it is considered that the LOAEL level for rats is 32 mg/kg bw/day and as a result the test material requires classification as STOT RE Category 2 in accordance with EU criteria and as defined in Annex VI, Regulation 1272/2008.