Registration Dossier

Administrative data

Description of key information

REPEATED DOSE TOXICITY: ORAL
28 Day NOEL for tall oil 1000 ppm male and female Sprague-Dawley rats
28 Day NOAEL for tall oil, compound with ethanolamine 300 mg/kg male and female Wistar rats
90 Day LOAEL for diethanolamine 20 mg/kg in F344 rats
90 Day LOAEL for diethanolamine 123 mg/kg in B6C3F1 mice
REPEATED DOSE TOXICITY: DERMAL
90 Day LOAEL for diethanolamine 32 mg/kg male and female F344 rats
90 Day LOAEL for diethanolamine 80 mg/kg male B6C3F1 mice

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
March 1987 to January 1988
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.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: ca. 6 weeks of age
- Weight at study initiation: Males had a mean initial bodyweight of 117 to 123 g; females had a mean initial bodyweight of 102 to 105 g.
- Housing: 5 animals per cage
- Diet (e.g. ad libitum): diet in pellet form was available ad libitum.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 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
Route of administration:
oral: drinking water
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: solutions were prepared in deionised water and 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:
Dose formulations were analysed by gas chromatography before and after administration to animals and found to be within 15 % of the theoretical values.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
Males: 0, 320, 630, 1250, 2500 or 5000 ppm; females: 0, 160, 320, 630, 1250 and 2500 ppm
Basis:
nominal in water
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 0, 630, 1250, 5000, and 10 000 ppm in the drinking water. All female rats in the two highest dose groups and 2 males in the 10 000 ppm group died before the end of the study.
Male and female rats had increased kidney weights, renal tubular cell necrosis, and decreased renal function; degeneration of the seminiferous tubules of the testis was noted in dosed males.
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 anaesthetized 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 anaesthetized 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, 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, 630, 1250 and 2500 ppm.
- 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.
Clinical signs:
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):
effects observed, treatment-related
Description (incidence and severity):
see below
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):
se 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:
Two males in the high dose (5000 ppm) group died before the end of the study. One female death seen 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 bodyweight gain. Based on water consumption and bodyweight data, average daily doses of the test material were estimated to range from about 25 to 440 mg/kg in males and about 15 to 240 mg/kg in females.
Summary data is provided in Table 1.

Clinical signs of toxicity included tremors, emaciation, abnormal posture, and rough hair coat in the two highest dose groups of each sex.

Administration of the test material produced a moderate, poorly regenerative, microcytic, normochromic anaemia in male and female rats (Table 2). Haematologic effects were dose-dependent and included decreases in erythrocyte and reticulocyte counts, haemoglobin concentration, haematocrit, MCV, and MCH. MCV was reduced in rats at all dose levels. Haematologic effects were not associated with microscopic changes in the femoral bone marrow.

No significant gross lesions attributable to the test material were found at necropsy. Dose-related increases in relative kidney weights were observed in males and females (Table 3). Kidney weight changes were accompanied by increases in the incidence and/or severity of nephropathy, renal tubular cell necrosis, or tubular mineralisation (Table 4). 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 characterised by eosinophilic tubular epithelial cells with pyknotic nuclei, frequently seen desquamated into the lumen of renal tubules. Mineralisation was observed as basophilic concretions within necrotic tubules which were present primarily along the outer stripe of the outer medulla. Mineralisation 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 toxicity. In the brain, microscopic change was observed in coronal sections of the medulla oblongata and consisted of bilaterally symmetrical areas of vacuolisation of the neuropil (Table 4). 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 (Table 4). There were no neurologic clinical signs that could be clearly attributed to these lesions

Decreases in testis and epididymis weights (Table 3) were associated microscopically with degeneration of seminiferous epithelium and with hypospermia. The testicular lesion 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 vacuolisation 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 of 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 (Table 3). 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.
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: A NOAEL was not achieved for the haematological changes or nephropathy.
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
LOAEL
Effect level:
160 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based upon haematological changes, nephropathy and effects on kidney weight.
Dose descriptor:
LOAEL
Effect level:
14 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based upon haematological changes, nephropathy and effects on kidney weight.
Dose descriptor:
LOAEL
Effect level:
320 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Based upon haematological changes, nephropathy and effects on kidney weight.
Dose descriptor:
LOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Based upon haematological changes, nephropathy and effects on kidney weight.
Critical effects observed:
not specified

Table 1 Summary of Survival, Weight Gain and Water Consumption

Sex

Dose

(ppm)

Survival

Mean Bodyweight

(g)

Final Weight Relative to Controls (%)

Average Water Consumption (mL/animal/day)

Estimated Test Material Consumed (mg/kg bw/day)

Initial

Final

Change

 

 

Male

0

320

630

1250

2500

5000

10/10

10/10

10/10

10/10

10/10

8/10

122

123

122

117

123

121

362

344

322

297

258

202

240

221

200

180

135

81

-

95

89

82

71

56

20.9

20.2

19.2

18.3

17.7

15.6

0

25

48

97

202

436

 

 

Female

0

160

320

630

1250

2500

10/10

9/10

10/10

10/10

10/10

10/10

102

105

103

105

102

104

222

211

201

200

187

167

120

106

98

95

85

63

-

95

91

90

84

75

15.5

14.9

16.9

15.2

15.8

13.9

0

14

32

57

124

242

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 (ppm)

0

160

320

630

1250

2500

5000

 

 

Male

RBC (10/µL)

HGB (g/dL)

HCT (%)

MCV (fL)

MCH (pg)

Reticulocytes (10/µL)

8.79

14.8

47.8

54

16.9

0.23

-

-

-

-

-

-

8.75

14.3*

46.1

53**

16.4**

0.23

8.20**

13.3**

42.5**

52**

16.2**

0.23

7.33**

11.6**

36.9**

50**

15.9**

0.24

6.40**

9.8**

31.4**

49**

15.3**

0.14**

5.71**

8.9**

27.8**

49**

15.5**

0.16**

 

 

Female

RBC (10/µL)

HGB (g/dL)

HCT (%)

MCV (fL)

MCH (pg)

Reticulocytes (10/µL)

8.40

15.1

47.3

56

17.9

0.17

8.51

15.2

47.0

55**

17.8*

0.16

7.84**

13.8**

42.3**

54**

17.7**

0.13**

7.56**

13.0**

39.7**

53**

17.2**

0.12*

6.78**

11.3**

34.4**

51**

16.7**

0.09**

6.43**

10.5**

31.2**

49**

16.3**

0.08**

-

-

-

-

-

-

Values given for the females at the 160 ppm dose level are for 9 animals.

Values given for the males at the 5000 ppm dose level are for 8 animals.

* = 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, Liver and Epididymis 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 (ppm)

0

160

320

630

1250

2500

5000

 

 

Male

Necropsy bodyweight

Kidney Weight

Relative kidney weight

Liver Weight

Relative liver weight

Right testis weight

Relative testis weight

Epididymis weight

Relative epididymis weight

366

1.29

3.54

15.09

41.28

1.49

4.08

0.426

1.17

-

-

-

-

-

-

-

-

-

339

1.34

3.94**

13.87

40.79

1.46

4.31

0.453

1.34**

326

1.30

3.99**

14.92

45.61**

1.47

4.50

0.392

1.20

302

1.21

3.98**

14.82**

48.90

1.27**

4.22

0.309**

1.02**

265

1.18

4.44**

14.18

53.27**

0.97**

3.64**

0.184**

0.68**

205

1.26

6.14**

11.59**

56.71**

0.54**

2.63**

0.134**

0.65**

 

 

Female

Necropsy bodyweight

Kidney Weight

Relative kidney weight

Liver Weight

Relative liver weight

218

0.66

3.03

6.08

27.86

208

0.86**

4.12

6.36

30.54

201

0.84**

4.21**

7.04**

35.09**

202

0.83*

4.12**

6.99**

34.52**

188

0.87**

4.63**

7.78**

41.41**

162

0.92**

5.67**

7.32**

45.26**

-

-

-

-

-

* = 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 Spinal Cord Lesions

Sex

Parameter

Dose (ppm)

0

160

320

630

1250

2500

5000

 

 

Male

Kidney

Nephropathy

Tubular epithelial necrosis

Tubular mineralisation

Brain, medulla

Demyelination

Spinal cord

Demyelination

6/10 (1.0)

0/10

0/10

0/10

0/10

-

-

-

-

-

2/10 (1.0)

0/10

0/10

0/10

0/10

2/10 (1.0)

0/10

0/10

0/10

0/10

3/10 (1.0)

0/10

1/10 (1.0)

0/10

0/10

6/10 (1.0)

0/10

10/10 (1.8)

10/10 (1.7)

10/10 (1.9)

10/10 (2.4)

10/10 (1.0)

10/10 (1.7)

10/10 (2.0)

10/10 (2.0)

 

 

Female

Kidney

Nephropathy

Tubular epithelial necrosis

Tubular mineralisation

Brain, medulla

Demyelination

Spinal cord

Demyelination

2/10 (1.0)

0/10

10/10 (1.3)

0/10

0/10

9/10 (1.0)

0/10

10/10 (2.0)

0/10

0/10

10/10 (1.5)

0/10

10/10 (2.5)

0/10

0/10

10/10 (1.4)

0/10

10/10 (3.0)

0/10

0/10

9/10 (1.0)

1/10 (1.0)

10/10 (2.4)

10/10 (1.5)

10/10 (1.0)

2/10 (1.0)

3/10 (1.0)

10/10 (1.7)

10/10 (1.9)

10/10 (1.9)

-

-

-

-

-

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 from groups of 10.

Conclusions:
A No-Observed-Adverse-Effect Level (NOAEL) was not achieved for the haematological changes or nephropathy. On this basis, it is considered that the LOAEL level for female rats is 160 ppm (14 mg/kg actual dose received) and for male rats is 320 ppm (25 mg/kg actual dose received) 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 408 under GLP conditions.

The test material was administered to male and female F344 rats for 13 weeks' duration in the animals’ drinking water.

Doses ranged from 160 to 5000 ppm in the drinking water (equivalent to daily doses of 25 to 440 mg/kg in males and 15 to 240 mg/kg in females).

Dose-dependent toxic effects due to exposure to the test material included haematological changes (a poorly regenerative, microcytic anaemia), as well as toxic responses in the kidney (increased weight, tubular necrosis, decreased renal function, and/or tubular mineralisation), brain and spinal cord (demyelination) and testis (degeneration of the seminiferous tubules).

A No-Observed-Adverse-Effect Level (NOAEL) was not achieved for the haematological changes or nephropathy. On this basis, it is considered that the LOAEL level for female rats is 160 ppm (14 mg/kg actual dose received) and for male rats is 320 ppm (25 mg/kg actual dose received) 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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
20 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
All studies were conducted under GLP conditions and were awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch (1997) based on the read across nature of the studies.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
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.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
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
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
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.
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.
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.
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.
Critical effects observed:
not specified

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.

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
32 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Both the key and supporting studies were conducted using a procedure equivalent to the standardised guideline OECD 411 under GLP conditions and as such were awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch (1997).

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
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.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
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
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
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.
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.
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.
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.
Critical effects observed:
not specified

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.

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
32
Study duration:
subchronic
Species:
rat
Quality of whole database:
Both the key and supporting studies were conducted using a procedure equivalent to the standardised guideline OECD 411 under GLP conditions and as such were awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch (1997).

Additional information

Repeated Dose Toxicity: Oral

The toxicity of the target substance was determined using read across to the two major metabolites, tall oil and diethanolamine (DEA). The metabolites were determined using TIMES rat liver S9 metabolism simulator as described in the Read Across Justification Document in Section 13.

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the read across material tall oil, the first source substance, in accordance with the standardised guideline OECD 422 under GLP conditions.

Four groups of 10 male and 10 female Sprague-Dawley rats received the test material via the diet at concentrations of 0, 1000, 5000 and 20 000 ppm. The males were dosed for at least 4 weeks, starting 2 weeks prior to mating. The females were dosed from 2 weeks prior to mating until at least day 6 of lactation.

The animals were monitored for clinical signs, bodyweight, food consumption, mating and litter performance. Haematology and clinical chemistry parameters were investigated; additionally, all animals were subjected to necropsy and histopathological investigations were carried out on animals in the control and 20 000 ppm dose groups.

At 20 000 ppm, in-life observations included decreased weight gain and food consumption in both sexes. Increased male liver weight following covariance analysis, and increases in bilirubin and alkaline phosphatase were noted in both sexes. In addition, small decreases were noted in adrenal gland weight in both sexes, and in albumin, white blood cell count and ovary weight in females; spleen weight and cholesterol were slightly increased in males.

At 5000 ppm, alkaline phosphatase levels in both sexes were increased. Female adrenal gland weight was reduced.

Under the conditions of this study, toxicity was exhibited at levels of 5000 and 20 000 ppm, but there were no clear effects of toxicity at 1000 ppm. Therefore the parental repeated dose NOEL was considered to be 1000 ppm.

 

The repeated dose toxicity of the read across material diethanolamine, the second source substance used for read across, was investigated in a procedure equivalent to the standardised guideline OECD 408 under GLP conditions.

The test material was administered to male and female F344 rats for 13 weeks' duration in the animals’ drinking water.

Doses ranged from 160 to 5000 ppm in the drinking water (equivalent to daily doses of 25 to 440 mg/kg in males and 15 to 240 mg/kg in females).

Dose-dependent toxic effects due to exposure to the test material included haematological changes (a poorly regenerative, microcytic anaemia), as well as toxic responses in the kidney (increased weight, tubular necrosis, decreased renal function, and/or tubular mineralisation), brain and spinal cord (demyelination) and testis (degeneration of the seminiferous tubules).

A No-Observed-Adverse-Effect Level (NOAEL) was not achieved for the haematological changes or nephropathy. On this basis, it is considered that the LOAEL level for female rats is 160 ppm (14 mg/kg actual dose received) and for male rats is 320 ppm (25 mg/kg actual dose received) 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.

 

The repeated dose toxicity of the read across material diethanolamine was further investigated in a procedure equivalent to the standardised guideline OECD 408 under GLP conditions in a second rodent species.

The test material was administered to male and female B6C3F1 mice for 13 weeks' duration in the animals’ drinking water.

Doses ranged from 630 to 10 000 ppm in the drinking water (equivalent to daily doses of 100 to 1700 mg/kg in males and 140 to 100 mg/kg in females).

Nephropathy and tubular necrosis were observed in males and degeneration of cardiac myocytes and hepatocellular necrosis were seen in males and females. Cytologic alteration in the submandibular salivary gland was noted in male and female mice. Hepatocyte cytologic alteration was also noted in all dosed groups of mice.

A No-Observed-Adverse-Effect Level (NOAEL) was not achieved for the hepatocellular cytological alterations. On this basis, it is considered that the LOAEL level for mice is 630 ppm; for female mice the LOAEL is 142 mg/kg actual dose received and for male mice it is 104 mg/kg actual dose received. In accordance with Annex VI, Regulation 1272/2008, the test material requires classification as STOT RE Category 2.

Finally, a combined repeated dose toxicity study with reproduction/developmental toxicity screening test is provided to assess the read across material tall oil, compound with ethanolamine in accordance with the standardised guideline OECD 422 under GLP conditions. This study is not used for weight of evidence only.

Three groups of 10 male and 10 female rats of the Crl:WI(Han) strain were dosed once daily, by oral gavage, with the test material at dose levels of 100, 300, or 1000 mg/kg/day. Another group of 10 male and 10 female rats were dosed with the vehicle (UHP water) following the same dosing regimen as the treated animals and were used as controls. The males were dosed for 14 days prior to pairing, during pairing and until the day prior to necropsy. The females were dosed for 14 days prior to pairing, during pairing and gestation, and up to and including Day 3 of lactation.

Clinical signs, body weights, food consumption and functional observations including grip strength and motor activity were recorded at pre-determined intervals. On Day 14 of dosing, blood samples were taken from 5 parental animals of each sex in all groups for clinical pathology evaluations. All animals were subjected to necropsy and tissues from control and high dose groups were microscopically examined.

There were no unscheduled deaths on the study and administration of the test material was well tolerated. Animals of both sexes given 1000 mg/kg/day showed slightly lower bodyweight gains than controls during the early part of the dosing period. Males given the test material at all dose levels had lower lymphocyte counts than controls and an increase in bone marrow adipocytes was seen in males at the highest dose level only. These findings were considered to be possibly treatment-related but the magnitude of change was small.

Under the conditions of this study, in the Wistar strain rat, the No Observed Adverse Effect Level (NOAEL) for male and female repeated-dose toxicology was considered to be 300 mg/kg/day.

 

In accordance with the general principles of Annex XI of Regulation (EC) 1907/2006 (REACH), it is considered scientifically justified to omit the sub-chronic repeated dose toxicity study on the tall oil component of the registered substance.

Sub-chronic repeated dose data is provided for diethanolamine in two species. This material is more toxic and in accordance with Annex VI, Regulation 1272/2008, requires classification as STOT RE Category 2.

As such, it is considered that further animal testing on this substance is unlikely to add substantial value to the dataset and is therefore not proposed by the registrant at this time.

 

Repeated Dose Toxicity: Inhalation

In accordance with the Column 2 adaptation of Annex VIII of Regulation (EC) 1907/2006 (REACH), it is considered justified to omit the repeated dose toxicity by the inhalation route study as specified in section 8.6.1. as testing by this route is inappropriate. Exposure via the inhalation route is not relevant due to the substance being an immobile paste with a low vapour pressure.

 

Repeated Dose Toxicity: Dermal

The repeated dose toxicity of the read across material diethanolamine was investigated in the key study 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.

 

Supporting information is provided in the form of further sub-chronic data in a second rodent species conducted with the read across material diethanolamine in a procedure equivalent to the standardised guideline OECD 411 under GLP conditions.

The test material was administered to male and female B6C3F1 mice for 13 weeks' duration, 5 days per week, to the shaved interscapular region. Doses ranged from 80 to 1250 mg/kg bw/day prepared in 95 % ethanol.

Early deaths were observed in the highest dose group (1250 mg/kg) and bodyweight gains were reduced in mice given the higher doses.

Skin lesions at the site of application included ulceration and inflammation, hyperkeratosis, and acanthosis. Animals exhibited cytological alterations in the liver and/or hepatocellular necrosis, renal tubular epithelial necrosis, and cardiac myocyte degeneration.

A No-Observed-Adverse-Effect Level (NOAEL) was not achieved for hepatocellular cytological alterations and acanthosis in the skin. On this basis, it is considered that the LOAEL level for mice is 80 mg/kg bw/day. In accordance with Annex VI, Regulation 1272/2008, the test material requires classification as STOT RE Category 2.

In accordance with the general principles of Annex XI of Regulation (EC) 1907/2006 (REACH), it is considered scientifically justified to omit the sub-chronic repeated dose toxicity study by the dermal route of exposure on the tall oil component of the registered substance. Sub-chronic repeated dose data is provided for diethanolamine in two species; this material is more toxic and in accordance with Annex VI, Regulation 1272/2008, requires classification as STOT RE Category 2.

As such, it is considered that further animal testing on this substance is unlikely to add substantial value to the dataset and is therefore not proposed by the registrant at this time.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
This endpoint is addressed on a weight of evidence basis using read across data to both tall oil and diethanolamine. This is based on the metabolism of the target substance to tall oil and diethanolamine (DEA) based on the TIMES S9 rat liver metabolism simulator as described in the Read Across Justification Document in Section 13. Based on the effects obsered in the 90 day repeat dose study on DEA, the target substance is classified as STOT RE 2. The 28 day repeat dose study is waived due to the availability of a 90 day study on one of the read across constituents and that the substance undergoes immediate disintegration and there are sufficient information on the cleavage products. This waiver is in line with Annex XIII documentation.
Two OECD 422 studies are provided to address the sub-acute repeated dose toxicity of tall oil; one conducted with tall oil and the other with tall oil, compound with ethanolamine. Sub-chronic repeated dose data is provided for diethanolamine in two species.
The sub-chronic study in the rat with diethanolamine was selected as key on the basis that this material is more toxic than the tall oil component of the registered substance and will therefore be responsible for the classification of the test material.
It is considered that these studies together are an accurate reflection of the total composition of the registered substance, Tall Oil, compound with diethanolamine.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
A data waiver has been submitted to address this endpoint.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
A data waiver has been submitted to address this endpoint.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Two studies are provided that address the sub-chronic toxicity of the read across material diethanolamine. The study in F344 rats was selected as key over the supporting study in B6C3F1 mice on the basis that the rat is the more commonly used rodent species.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Two studies are provided that address the sub-chronic toxicity of the read across material diethanolamine. The study in F344 rats was selected as key over the supporting study in B6C3F1 mice on the basis that the rat is the more commonly used rodent species.

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: other; digestive: liver; urogenital: kidneys

Repeated dose toxicity: dermal - systemic effects (target organ) cardiovascular / hematological: other

Justification for classification or non-classification

In accordance with Annex VI, Regulation 1272/2008, diethanolamine requires classification as STOT RE Category 2. As such, on a precautionary basis, the registered material will also be classified as STOT RE Category 2.