Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

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.

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 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
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. 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

Administration / exposure

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
Doses / concentrations
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.

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 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.

Results and discussion

Results of examinations

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.

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 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.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.