Registration Dossier

Administrative data

Description of key information

The substance induced significant decrease in body weight and food consumption by both the oral and inhalation routes. 
Similar effects were observed for both exposure routes: altered organ organ weights (liver, kidney, brain, adrenals, testes, epididymis, thymus), altered haematological parameters (in particular red blood cells and hemoglobin levels). In addition, in the oral study, the prostate and seminal vesicles weights were found lower in all treated groups, while the weight of lungs, thyroid and hypophysis were increased. White blood cells and lymphocytes were also found decreased.
Histopathological examinations showed effects in liver, thymus, male and femal reproductive organs. In males, decreased activity of Inhibin B in the 2 higher dose groups of the 28-day inhalation study was consistent with testicular damage.
The liver effects may represent an adaptative response as evidenced by increased liver weight, liver enlargement, hepatocytes hypertrophy or swelling, decreased cholesterol levels and increased total proteins. In the inhalation study, analysis of thyroid hormones showed a decrease in T3 at the high dose, and increased levels of T4 in all dose groups, consistent with the inhibition of 5'-deiodinase activity observed in vitro in rat hepatocytes.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1997 ?
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Only the results (mean group data) are available (translation from a report in Japanese). The material and methods information was not available.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
not specified
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
No data available

ENVIRONMENTAL CONDITIONS
No data available

IN-LIFE DATES: No data available
Route of administration:
oral: gavage
Vehicle:
not specified
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
No data available

VEHICLE
No data available
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
1, 3, 10, and 20 mg/kg
Basis:

No. of animals per sex per dose:
6 per sex and per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale, rationale for animal assignment: no data available
- Rationale for selecting satellite groups: the satellite groups were daily administered the test item at 10 or 20 mg/kg bw for 28 days.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale: no data available
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule, cage side observations checked: no data available

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: no data available

BODY WEIGHT: Yes
- Time schedule for examinations: once on the day prior to the start of the administration; once on the first day of administration and then once every 2-3 days during the administration period (28 days) for all treated groups; and once on the first day of recovery and then once every 2-3 days during the recovery periods (14 days) for the control, 10-mg/kg and 20-mg/kg groups

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; once on the first day of administration and then once every 3-4 days during the administration period (28 days) for all treated groups; and once on the fourth day of recovery and then once every 3-4 days during the recovery periods (14 days) for the control, 10-mg/kg and 20-mg/kg groups
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood, anaesthetic used for blood collection, animals fasted, how many animals: no data available
- Parameters checked: red blood cells (RBC), white blood cells (WBC), haemoglobin (Hb), haematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count, reticulocyte count, coagulation (prothrombin time (PT), activated partial thromboplastin time (APTT)), differential leukocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood, animals fasted, how many animals: no data available
- Parameters checked: glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), alkaline phosphatase (ALP), cholinesterase (ChE), gamma-glutamyl transpeptidase (γ-GTP), total cholesterol (T-Cho), triglycerides (TG), glucose, total protein, albumin, globulin, albumin/globulin ratio, blood urea nitrogen (BUN), creatinine, total bilirubin (T-Bil), calcium (Ca), inorganic phosphorus (IP), sodium (Na), potassium (K), chloride (Cl).

URINALYSIS: Yes
- Time schedule for collection of urine, metabolism cages used for collection of urine, animals fasted: no data available
- Parameters checked: volume, colour, pH, protein, ketones, bilirubin, occult blood, glucose, urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Adrenal glands
Brain
Cecum
Colon
Duodenum
Epididymides
Heart
Hypophysis
Ileum
Incisors
Jejunum
Kidneys
Liver
Lymph nodes - mesenteric
Mammary gland
Oral cavity
Ovary
Pituitary gland
Prostate
Rectum
Seminal vesicles
Skin
Spleen
Stomach - forestomach, glandular stomach
Testis
Thymus
Thyroid gland
Uterus
Vagina
All gross lesions
Other examinations:
ORGAN WEIGHTS (absolute and relative):
Adrenal glands
Brain
Epididymides
Hypophysis
Kidney
Liver
Ovary
Prostate
Seminal vesicle
Spleen
Testis
Thymus
Thyroid gland
Uterus
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Mortality:
mortality observed, treatment-related
Description (incidence):
see in "Details on results"
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY (Table 1 in "Any other information on results incl. tables")
The administration of the test item caused no death during the administration period. Further, signs of neurosis were noted in a preliminary test.
Salivation was seen in almost all male and female rats from vehicle control and treated groups, regardless of the dose level administered. Although this clinical sign was observed in control animals, it was considered to be the effect of the test substance. This effect was not observed during the recovery period.
Some clinical signs were described in few animals treated with the test item, regardless of the dose level: e.g., alopecia, coarse and hard coat of hair, dirt around the anus and the hypogastric region, emaciation; all these effects remained in rare animals of both genders during the recovery period.
Fallen spontaneous movement was mainly seen in males and females rats treated with the dose levels of 10 and 20 mg/kg bw/day and it was still observed during the recovery period in the major part of treated animals. 
New clinical signs appeared in few animals of both sexes during the recovery period: e.g., whitening of the tails, scab formation.

BODY WEIGHT AND WEIGHT GAIN (Table 2 in "Any other information on results incl. tables")
During the administration period, males and females treated with dose levels equal or higher than 3 mg/kg bw/day showed either reduced body weight or inhibited body weight gain. Moreover, males and females treated with a dose of 1 mg/kg bw/day displayed an inhibited body weight gain or such a tendency. Although a tendency towards recovery was noted in males and females treated with dose levels of 10 or 20 mg/kg bw/day, yet the low body weights remained during the recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE (Table 2 in "Any other information on results incl. tables")
During the administration period, animals treated with dose levels equal or higher than 3 mg/kg bw/day showed reduced food consumption. Although a tendency towards recovery was noted in animals treated with dose levels of 10 or 20 mg/kg bw/day, yet the low feeding amount remained in males while an increase in feeding amount was observed in females treated with dose level of 20 mg/kg bw/day.

HAEMATOLOGY (Table 3 in "Any other information on results incl. tables")
At the end of the administration period, almost all significant haematological impairments were seen at a dose level equal and/or higher than 3 mg/kg bw/day: decrease in red blood cell (RBC) number in all females; decrease in white blood cell (WBC) number, haemoglobin (Hb) and haematocrit (Ht) concentration, average volume of red blood cells (MCV), average amount of red blood cell haemoglobin (MCH) in all treated males and females; prolonged prothrombin time (PT) and prolonged time of the activated portion of thromboplastin (APTT) in males and females. 
The average density of red blood cell haemoglobin (MCHC) was mainly decreased in females treated with the test item. Similarly, only females treated with 10 or 20 mg/kg bw/day displayed a decrease in lymphocyte and monocyte percentages and an increase in neutrophil percentage in the segmentation nucleus. 
During the recovery period, the major part of the haematological changes abovementioned remained: e.g., prolonged time of APTT and decreased MCH in males; MCV decrease in females; decrease in WBC number, Hb and Ht concentration, MCHC, lymphocyte percentages in males and females. In addition, new haematological changes appeared in animals of both sexes during the recovery period: i.e., increase in reticulocyte percentage.
All these changes were considered to be caused by the influence of the test substance.

CLINICAL CHEMISTRY (Table 4 in "Remarks on results including tables and figures")
Regardless of the dose level administered, all males and females displayed a decrease in total cholesterol and albumin/globulin ratio at the end of the administration period. Moreover, from the dose level of 3 mg/kg bw/day and/or above, animals of both sexes showed an increase in total protein, gamma-GTP, total bilirubin, and GPT. Furthermore, at the end of the administration period, increased BUN and decreased inorganic phosphorus levels were seen in males at 10 and 20 mg/kg bw/day; while decreased ChE and chloride levels and increased potassium levels were measured in females at 10 and/or 20 mg/kg bw/day.
During the recovery period, some biochemistry changes remained: increased GPT in males; decreased ChE and increased gamma-GTP in males and females. In addition, new biochemistry changes appeared in males and females during the recovery period: decrease in creatinine in males and females; decrease in inorganic phosphorus and increase in TG, sodium and calcium in females. 
All these changes were considered to be caused by the influence of the test substance.

URINALYSIS
Uroscopy showed a tendency towards increased albumin in urine in males treated with 10 mg/kg bw/day and more and females treated with a dose level of 20 mg/kg bw/day. At the end of the period of recovery various changes were found to remain.

ORGAN WEIGHT (Table 5 in "Remarks on results including tables and figures")
After 28 days of treatment, the weight of various organs was significantly altered in treated animals of both sexes. When expressed as organ/body weight ratio, the weight of thymus, prostate, seminal vesicles, epididymis, and testes significantly decreased in treated males, while the weight of brain, liver, kidneys, adrenals, hypophysis, and thyroid significantly increased in the same treated groups. Overall, treated females displayed significant decreases in thymus weight and significant increases in brain, liver, kidney, thyroid, hypophysis, and uterus weights (i.e., when expressed as organ/body weight ratio). 
After 14 days of recovery, the relative weight of almost all organs abovementioned remained significantly altered. When expressed as organ/body weight ratio, epididymis, prostate, seminal vesicles and testes weights were still significantly decreased, and the weight of spleen, liver, kidney, brain, thyroid, hypophysis, lungs, thymus, and adrenals significantly increased in males at the end of the recovery period. In females, the weight of brain, adrenals, spleen, liver, kidney, hypophysis weights was still increased and the decrease in thymus relative weight remained. 
All these changes were considered to be caused by the influence of the test substance.

GROSS PATHOLOGY (Table 6 in "Attached background material")
After 28 days of administration, the main gross lesions observed were localised in the liver from males and females of groups treated with 3, 10 and 20 mg/kg bw/day (i.e., accentuated lobular pattern with whitish margin and enlargement). Furthermore, a reduction in thymus size and whitish spots in mesenteric lymph nodes were also seen in all animals treated with dose levels of 10 and/or 20 mg/kg bw/day. In addition, all treated males from dose groups 3, 10 and 20 mg/kg bw/day showed a size reduction of testes, epididymis, prostate and seminal vesicles. A smaller seminal vesicle and prostate was also noticed in 3 males from the group administered 1 mg/kg bw/day.
After 14 days of recovery, males still showed hepatic lesions and size reduction of testes, epididymis, prostate and seminal vesicles, regardless of the dose level. Gross lesions in the liver remained in all females treated with the dose levels of 10 and/or 20 mg/kg bw/day.

HISTOPATHOLOGY: NON-NEOPLASTIC (Table 6 in "Attached background material")
After 28 days of treatment, hepatic, spleen, renal, and thymus lesions were seen in males and females treated with the test item, notably at the dose levels of 10 and 20 mg/kg bw/day (e.g., swollen hepatic cells, hemosiderin deposition in the spleen, atrophy of thymus). Moreover, atrophy of seminal vesicles, testes, epididymis, and prostate, and congestion of lymph nodes was observed in all males from the dose level of 3 mg/kg bw/day. In addition, histopathological lesions of ovaries, uterus, vagina and mammary glands were described in all females exposed to the test substance, regardless of the dose level (i.e., decreased copora lutea and increased follicular cyst in ovaries, tall columnar epithelial cells in uterus, mucification in vagina, hyperplasia of mammary glands).  
After the 14-day recovery period, the findings in liver, spleen, kidneys, ovaries, mammary glands, and male reproductive organs were still present.
Dose descriptor:
NOEL
Effect level:
< 1 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEL
Effect level:
1 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified

Table 1: Significant clinical signs after 28-day treatment and 14-day recovery

Gender

After 28-day treatment

After 14-day recovery

Males

Females

Males

Females

Dose (mg/kg bw/day)

0

1

3

10

20

0

1

3

10

20

0

10

20

0

10

20

Salivation

9/12

6/6

6/6

12/12

12/12

8/12

6/6

6/6

12/12

12/12

0/6

0/6

0/6

0/6

0/6

0/6

Decreas. spontan. locomotion

0/12

0/6

3/6

12/12

12/12

0/12

0/6

1/6

12/12

12/12

0/6

1/6

4/6

0/6

3/6

4/6

Decreas. respirat. rate

0/12

0/6

0/6

0/12

3/12

0/12

0/6

0/6

0/12

3/12

0/6

0/6

0/6

0/6

0/6

0/6

Emaciation

0/12

0/6

0/6

2/12

1/12

0/12

0/6

0/6

4/12

1/12

0/6

0/6

0/6

0/6

1/6

1/6

Unkempt hair

0/12

0/6

1/6

3/12

3/12

0/12

2/6

0/6

1/12

2/12

0/6

0/6

2/6

0/6

1/6

1/6

Loss and discolour. up. incisor

-

-

-

-

-

0/12

0/6

0/6

0/12

1/12

-

-

-

0/6

0/6

0/6

Loss and discolour. low. incisor

-

-

-

-

-

0/12

0/6

0/6

0/12

1/12

-

-

-

0/6

0/6

2/6

Staining around nose and mouth

0/12

3/6

2/6

4/12

5/12

0/12

3/6

3/6

2/12

2/12

0/6

0/6

0/6

0/6

0/6

0/6

Staining hair

-

-

-

-

-

0/12

2/6

0/6

0/12

0/12

-

-

-

0/6

0/6

0/6

Staining lower abdomen

0/12

0/6

0/6

0/12

2/12

0/12

0/6

1/6

1/12

1/12

0/6

0/6

1/6

0/6

0/6

0/6

Staining around anus

0/12

0/6

2/6

0/12

1/12

0/12

0/6

2/6

1/12

0/12

0/6

0/6

0/6

0/6

1/6

0/6

Soft stool

0/12

0/6

0/6

2/12

3/12

0/12

0/6

0/6

4/12

7/12

0/6

0/6

0/6

0/6

0/6

0/6

Loss of hair

0/12

2/6

0/6

1/12

4/12

0/12

1/6

1/6

1/12

3/12

0/6

1/6

3/6

0/6

1/6

0/6

 

Table 2: Body weight and food consumption after 28-day treatment and 14-day recovery

Gender

After 28-day treatment

After 14-day recovery

Males

Females

Males

Females

Dose (mg/kg bw/day)

0

1

3

10

20

0

1

3

10

20

0

10

20

0

10

20

Body weight (g)

344.2

302.0*

193.5*

157.6*

157.8*

210.7

190.5

161.4*

138.0*

135.8*

412.3

208.7*

193.2*

228.5

174.3*

175.1*

Food consumption

(g/rat/day)

19.4

19.7

10.0*

7.7*

8.1*

12.8

11.1

8.3*

7.0*

6.7*

27.2

19.6*

18.6*

17.7

16.7

21.2*

* = statistically significant

 

Table 3: Summary of significant haematology results after 28-day treatment and 14-day recovery

Gender

After 28-day treatment

After 14-day recovery

Males

Females

Males

Females

Dose (mg/kg bw/day)

0

1

3

10

20

0

1

3

10

20

0

10

20

0

10

20

RBC (x 104/mm3)

747

741

714

690

704

776

775

716*

682*

680*

789

686*

660*

794

714*

688*

WBC (x10²/mm3)

101

95

66*

57*

57*

111

99

60*

58*

67*

132

67*

69*

103

53*

55*

Hb (g/dL)

14.7

14.4

13.5*

12.2*

12.5*

15.7

14.9

13.3*

12.4*

12.2*

15.3

12.7*

11.8*

15.1

13.5*

12.9*

Ht (%)

42.3

42.4

38.9*

36.2*

36.6*

43.9

43.0

39.2*

36.4*

35.8*

43.3

37.2*

34.5*

42.1

39.8

38.7*

MCV (µm3)

56.8

57.2

54.5

52.5*

52.1*

56.6

55.5

54.8

53.3*

52.7*

54.9

54.2

52.4

53.0

55.7*

56.3*

MCH (pg)

19.8

19.5

19.0

17.7*

17.8*

20.3

19.2*

18.7*

18.2*

18.0*

19.4

18.5

17.9*

19.0

18.9

18.7

MCHC (%)

34.9

34.0

34.8

33.8*

34.3

35.8

34.6*

34.0*

34.1*

34.1*

35.4

34.1*

34.2*

35.7

33.9*

33.3*

Reticulo (%)

27

21

20

25

18

24

25

25

21

22

26

76*

48

24

56*

72*

PT (sec)

11.5

12.3

12.0

12.9*

12.3*

11.5

12.3

12.4

12.4

13.0*

12.2

11.5

11.7

11.7

11.4

11.5

APTT (sec)

22.3

31.5*

31.6*

29.5*

27.8

20.7

28.4*

29.6*

31.3*

27.4*

23.8

17.9*

18.3*

19.7

18.6

19.8

Leukocyte N-seg (%)

17.7

16.1

20.7

25.2

25.0

9.9

13.3

17.8

25.9*

22.8*

12.3

25.5*

31.7*

14.1

28.8*

29.7*

Lymph

80.8

83.3

78.3

74.2

73.8

88.7

85.4

81.6

73.4*

76.7*

86.8

73.3*

67.0*

85.2

69.2*

68.7*

Mono

0.3

0.1

0.3

0.3

0.3

0.5

0.3

0.2

0.0*

0.0*

0.1

0.2

0.2

0.0

0.1

0.3

* = statistically significant

Conclusions:
Based on the results of this study, a no-observed-effect-level (NOEL) was estimated to be under 1 mg/kg bw/day for the test substance.
Target organs identified from the dose level of 1 mg/kg bw/day were the liver, kidneys and reproductive organs.
Executive summary:

The purpose of this 28-day oral toxicity study was to evaluate the range of toxicity and to indicate potential target organs associated with the exposure of the test substance when administered to rats by gavage daily for 28 days. In order to assess the reversibility of any treatment-related findings, satellite groups of animals were maintained for a subsequent recovery period of 14 days without treatment.

 

Groups of 6 male and 6 female Sprague-Dawley rats were exposed to the test substance at dose levels of 0 vehicle control), 1, 3, 10, and 20 mg/kg body weight (bw)/day. Additional satellite groups of 6 males and 6 females were treated with dose levels of 10 and 20 mg/kg bw/day for 28 days and then kept for a 14-day period to investigate the potential for recovery. Mortality, clinical signs, body weight, food consumption, water consumption, ophthalmoscopic examinations, clinical laboratory investigations, organ weights, macroscopic and microscopic findings were recorded.

 

There was no case of death caused by the administration of the test substance during the entire administration period. Abnormal symptoms, decreases or inhibition in body weight gain and low values of feed consumption were found among the groups exposed daily to the test substance.

 

Further, males and females administered with 1 mg/kg bw/day test substance or more developed organ lesions in the liver, spleen and reproductive organ during the administration period. From the dose level of 1 mg/kg bw/day, almost all of the changes found through the clinical biochemistry and haematological examinations were considered to have been caused by hepato-bile duct troubles and renal impairments. The haematological examinations showed evidence of anaemia and lymphocytic alterations and further revealed changes in the mammary glands, mesenteric glands and spleen. Moreover, the remarkable impact on the reproductive organs and teeth, that is, changes (including relative organ weight and histopathological findings) in testes, epididymis, seminal vesicles and prostate glands in case of males and changes in ovary, uterus, vagina and mammary glands in case of females, were considered as specific changes caused by the test substance, besides the changes in the pituitary glands/hypophysis depending on the dose administered.

 

Following the recovery period, abnormal symptoms and abnormality in body weight and food consumption remained. Moreover, at the end of the recovery period various changes were still found in the clinical biochemistry and haematological examinations, uroscopy analysis, through measurement of organ weights and in the pathological inspection.

  

Based on the results of this study, the no-observed-effect-level (NOEL) was estimated to be below 1 mg/kg bw/day. Target organs identified from the dose level of 1 mg/kg bw/day were the liver, spleen and reproductive organs.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
1 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Despite missing details on methodology due to partially translated report, the study seemed well-conducted and provided detailed results.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14-FEB-2007 to 03-DEC-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This GLP-compliant study was conducted according to OECD guideline 412.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: HsdHan (SPF)
- Source: Harlan Netherlands B.V. / Postbus 6174, NL-5960 AD Horst / THE NETHERLANDS
- Age at study initiation: 7 to 8 weeks for males; 9 to 10 weeks for females
- Weight at study initiation: the weight variation in animals entering the study did not exceed ± 7% of the mean weight of the corresponding sex.
- Fasting period before study: not applicable
- Housing: animals of the same sex were housed in groups of up to 5 in Makrolon type-IV cages with wire mesh tops and standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz / SWITZERLAND).
- Diet: ad libitum (pelleted standard Kliba-Nafag 3433 rat maintenance diet, batch no. 80/06), except during the daily exposure periods and during ca. 16 h prior to clinical laboratory investigations
- Water: ad libitum (community tap-water from Füllinsdorf, chlorinated to ca. 0.5 ppm), except during the daily exposure periods
- Acclimation period: 15 days, under laboratory conditions, after clinical health examination

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 30 to 70%
- Air changes: 10 to 15 changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From 21-FEB-2007 to 03-MAY-2007
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: compressed filtered air
Remarks on MMAD:
MMAD / GSD: Not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: inhalation by whole-body vapour exposure was performed in sealed chambers used for group isolation. These chambers were constructed of stainless steel. Glass doors were equipped with silicone rubber seals. The data of the exposure chambers are summarised in Table 1 below.
- Method of holding animals in test chamber: animals were accustomed to the whole-body inhalation chambers and the exposure conditions for 3 daily periods of ca. 1, 3 and 6 hours, respectively.
- Source and rate of air: the oxygen content was equal to 20.6%.
- Method of conditioning air: no data available
- System of generating particulates/aerosols: for group 2 (ca. 0.2 ppm), gas bags were prepared at a concentration that was defined during technical trials. From the gas bags, the vapour was transferred with a gas pump to a pipe and was then diluted with 420 L/min filtered air to the inlet duct of the exposure chamber.
For groups 3 (ca. 0.632 ppm) and 4 (ca. 2 ppm), the test substance was pumped at a specific rate into a round bottomed glass flask to be vaporised. Compressed air (40 L/min) was supplied into the glass flasks and allowed the liquid to equilibrate to the temperature of the warm walls of the container. The vapour produced then was passed through a pipe and was then diluted with 380 L/min of filtered air to the inlet duct of the exposure chamber.
Gas bags and flasks containing the test substance were kept at room temperature.
- Temperature, humidity, pressure in air chamber: ca. 24.5°C; ca. 33.4%; negative pressure of ca. 2 mm H2O
- Air flow rate: no data available
- Air change rate: 10 to 15 air changes per hour
- Method of particle size determination: not applicable
- Treatment of exhaust air: no data available

TEST ATMOSPHERE
- Brief description of analytical method used: The test item vapour was measured by on-line gas chromatography (GC).
- Samples taken from breathing zone: yes

VEHICLE: not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chamber concentration and stability of the test substance over the duration of ca. 6 hours was determined on at least two occasions prior to the start of the animal exposures. The test item was generated in the chambers at nominal concentrations of 0.2, 0.632 / 0.4 and 2 / 0.8 ppm and measured by on-line gas chromatography (GC).
During the treatment period, the concentration of the test substance in the chamber was determined daily, at least 5 times each hour of exposure. Start of recording was after the chamber equilibration time T90 (i.e. 90% equilibration time), and continued until pump off.
The method of analysis corresponded to the following:
- Column: DB-5 (No. 163755)
- Oven: 100°C, isothermal
- Runtime: 3 min
- Injector: 250°C
- Detector: 250°C
- Carrier gas: helium
- Sample loop: ca. 1700 µL
- Retention time: ca. 1.3
Duration of treatment / exposure:
4 consecutive weeks
Due to signs of overt toxicity (body weight loss, emaciation), the vapour concentrations for Groups 3 and 4 were lowered after 12 exposures, starting on Day 13 of exposure (exposures 13 to 20, phase 2).
Frequency of treatment:
6 hours/day and 5 days/week
Remarks:
Doses / Concentrations:
0.2, 0.632 and 2 ppm during phase 1 (exposure days 1 to 12) ; i.e., 0.0045, 0.01432 and 0.0453 mg/L
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.198, 0.635, 1.99 ppm during phase 1 (exposure days 1 to 12) ; i.e., 0.00448, 0.01438, and 0.045078 mg/L
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0.2, 0.4 and 0.8 ppm during phase 2 (exposure days 13 to 20); i.e., 0.0045, 0.0091, and 0.0181 mg/L
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.198, 0.434, 0.78 ppm during phase 2 (exposure days 13 to 20); i.e., 0.00448, 0.009831, and 0.0177 mg/L
Basis:
analytical conc.
No. of animals per sex per dose:
5 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: vapour concentrations were chosen on the basis of the results of the 5-day dose range finding inhalation toxicity study in the rat (see ESR entitled "I-(CF2)6-I - Repeat. dose tox.: 5-day inhal. - K1 2008RCC").
- Due to signs of overt toxicity (body weight loss, emaciation), the vapour concentrations for Groups 3 and 4 were lowered starting on Day 13 of exposure.

- Rationale for animal assignment (if not random): not applicable
- Rationale for selecting satellite groups, post-exposure recovery period in satellite groups: in order to assess the reversibility of any treatment-related findings satellite groups of animals were maintained for a subsequent period of 4 weeks without treatment.
- Section schedule rationale (if not random): not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: all animals were observed for mortality/morbidity twice daily, once prior to and once following exposure, during the treatment period and at least once daily on non-exposure days of treatment period and during the acclimatisation period and recovery periods.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical signs were recorded on the first day of the acclimatisation period. During the exposure period, clinical signs were observed twice daily, prior to and following exposure. On non-exposure days of the treatment period and during the recovery period, clinical signs were observed once daily. During exposure, only grossly abnormal signs were observed, as the animals were housed in the whole-body inhalation chambers. Observations included, but were not limited to: changes in the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern.

BODY WEIGHT: Yes
- Time schedule for examinations: each animal was weighed on the first day of the acclimatisation period (used for randomised), and thereafter at least once weekly during the acclimatisation, treatment and recovery periods.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; food consumption was measured per cage of 5 animals once weekly during the acclimatisation, treatment and recovery periods.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: water consumption was measured per cage of 5 animals at least twice weekly during the acclimatisation, treatment and recovery periods.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations, dose groups that were examined: ophthalmoscopic examinations was performed in all animals before treatment (during acclimatisation [23-FEB-2007]) and in all surviving animals in week 4 of the treatment period [29-MAR-2007] and in week 4 of the recovery period [27-APR-2007]. Observations were performed after instillation of a mydriaticum, using a Heine Bifocal Type Miroflex ophthalmoscope (Eisenhut Vet. AG, 4123 Allschwil / SWITZERLAND).

HAEMATOLOGY: Yes
- Time schedule for collection of blood, how many animals: blood samples from the retro-orbital plexus were collected for haematology from all animals of all groups under isofluorane (Forene) anaesthesia before necropsy. The samples were collected early in the working day to reduce biological variation caused by circadian rhythm.
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes; the animals were fasted in metabolism cages for ca. 16 hours before blood sampling, but water was provided ad libitum.
- Parameters checked: erythrocyte count (RBC), haemoglobin (HB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocyte count, red blood cell morphology (red cell volume distribution width (RDW), haemoglobin concentration distribution width), reticulocyte maturity index, platelet count, total leukocyte count (WBC), differential leukocyte count, coagulation (prothrombin time (PT), activated partial thromboplastin time (PTT))

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood, how many animals: blood samples from the retro-orbital plexus were collected for clinical biochemistry from all animals of all groups under isofluorane (Forene) anaesthesia before necropsy. The samples were collected early in the working day to reduce biological variation caused by circadian rhythm.
- Animals fasted: Yes; the animals were fasted in metabolism cages for ca. 16 hours before blood sampling, but water was provided ad libitum.
- Parameters checked: glucose, total cholesterol, creatinine, phospholipids, urea, total bilirubin, triglycerides, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase (LDH), glutamate dehydrogenase, creatinine kinase, gamma-glutamyl-transferase, chloride, calcium, sodium, potassium, total protein, phosphorus, albumin, globulin, albumin/globulin ratio.
- Additional investigations: alpha-glutathione S-transferase (alpha-GST) (by immuno assay), inhibin B (by ELISA), tri-iodothryronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH), testosterone by immunoassay.

URINALYSIS: Yes
- Time schedule for collection of urine: sampling during the 16-h period before blood sampling
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: volume, relative density, osmolality, colour, appearance, pH, protein, glucose, ketone, nitrite, bilirubin, erythrocytes, urobilinogen, leukocytes

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; all animals of all groups were necropsied. The surviving animals were fasted overnight before necropsy, but water was provided. All animals were anaesthetised by an intraperitoneal injection of sodium pentobarbitone and sacrificed by exsanguination.
HISTOPATHOLOGY: Yes; samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution unless otherwise stated. Lungs were instilled with the fixative at a hydrostatic pressure of 30 cm.
Adrenal glands
Aorta
Brain (cerebrum, cerebellum, brain stem)
Cecum
Colon
Duodenum
Epididymides (fixed in Bouin's solution)
Oesophagus
Extraorbital lacrimal glands
Eyes with optic nerves (fixed in Davidson's solution)
Femur, including joint
Harderian glands (fixed in Davidson's solution)
Heart
Ileum
Jejunum
Kidneys
Larynx (three transversal sections, at least levels II, III and IV)
Liver
Lungs
Lymph nodes - bronchial, mandibular, mesenteric
Mammary gland area
Nasal cavities (infused with formalin) with "paranasal sinuses" (Levels I to IV)
Nasopharyngeal duct and pharynx (1 longitudinal section)
Ovaries
Pancreas
Pituitary gland
Prostate
Rectum
Salivary glands - mandibular, sublingual
Sciatic nerve
Seminal vesicles (fixed in Bouin's solution)
Skeletal muscle (thigh region)
Skin
Spinal cord - cervical, midthoracic, lumbar
Spleen
Sternum with bone marrow
Stomach
Testes (fixed in Bouin's solution)
Thymus
Thyroid gland with parathyroid gland
Tongue
Trachea (one transversal section below larynx)
Tracheal bifurcation, carina and mainstem bronchi
Urinary bladder (infused with formalin)
Uterus with uterine cervix
Vagina
All gross lesions
Other examinations:
ORGAN WEIGHTS: the following organ weights were recorded at the scheduled dates of necropsy. Organ to body weight and organ to brain weight ratios were calculated. Paired organs were weighed together.
Adrenal glands
Brain
Epididymides
Heart
Kidneys
Liver
Lungs
Ovaries
Spleen
Testes
Thymus
Uterus

ENZYMATIC ASSAY:
It was hypothesised that the test substance could act as an inhibitor of the (hepatic) deiodinase leading to potential accumulation of T4 and concomitant decrease in T3. This suggested mode of action was investigated by determination of in vitro conversion of T4/T3 catalysed by rat and human liver microsomes in presence of the test substance.
Rat (Wistar) liver microsomes were prepared by RCC and tested for P-450 content and stored at -80°C until use. Human liver microsomes were obtained from Chemie Brunschwig AG (Basel, SWITZERLAND) and stored at -80°C until use.
Activity of 5’-deiodinase was determined by release of acid soluble iodide [125 I-] from 3,3’,5’-[125 I]-triiodo-L-thyronine ([125 I]-reverse T3) upon incubation with liver microsomes. Test item concentrations from 0 up to 1 mM were incubated with liver microsomes (4.4 µg protein) in a final volume of 100 µL of 0.1 M potassium hydrogen phosphate buffer/0.01 EDTA (pH 7.0), 1 mM DTT, 1 µM freshly prepared reverse T3 and [125 I]-reverse T3 (13.5 fmol/assay) at 37°C for 20 min. The reactions were stopped by addition of 50 µL 6-propyl-2-thiouracil (PTU) at 10 µM/8% BSA (50:50, v/v). The proteins were precipitated by addition of 400 µL 10% trichloroacetic acid and centrifugation at 3000 g for 15 min at 4°C. Released acid soluble iodide [125 I-] in the supernatant was determined by gamma-counting following purification by cation exchange chromatography.
A known inhibitor of 5’-deiodinase, PTU at 3-4 µM, was used as a positive control.

To test linearity of 5’-deiodinase activity rat liver microsomes were incubated with [125 I]-reverse T3 for 10, 20 and 40 min at protein concentrations of 0, 11, 22, 44, 88 and 176 µg/mL. 5’-deiodinase activity of rat liver microsomes was proportional to the incubation time and to the protein concentration up to 40 min at 44 µg protein/mL, up to 88 µg protein/mL at an incubation time of 20 min and up to 176 µg protein/mL at an incubation time of 10 min. Therefore 5’-deiodinase activity was investigated at a protein concentration of 44 µg/mL and an incubation time of 20 min, and with test item concentrations of 0, 1, 3, 7, 15, 63, 250 and 1000 µM.

% deiodination = ((sample cpm – blank cpm) x 100)/total cpm
where total cpm = cpm in incubation

Activity of 5’-deiodinase: % deiodination/protein concentration per assay/incubation time [% deiodination/ng protein/min]
Statistics:
The Dunnett test (many-to-one t-test) based on a pooled variance estimate was used to analyse body weight, organ weights and food and water consumption and clinical laboratory parameters.
The Steel test (many-one rank test) was applied instead of the Dunnett test for those clinical laboratory parameters that could not be assumed to follow a normal distribution.
The Fisher's exact test was applied to macroscopic findings and ophthalmoscopy data where appropriate.
Group means and standard deviations were calculated for continuous data.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no unscheduled deaths during the course of this study. However, in group 2, several females were emaciated towards the end of the treatment period. In group 4, the majority of animals of both genders were emaciated towards the end of the treatment period. The majority of animals of both genders from group 4 were still emaciated during the beginning of the recovery period. No further clinical signs were considered to be related to exposure to the test substance.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were no unscheduled deaths during the course of this study. However, in group 2, several females were emaciated towards the end of the treatment period. In group 4, the majority of animals of both genders were emaciated towards the end of the treatment period. The majority of animals of both genders from group 4 were still emaciated during the beginning of the recovery period. No further clinical signs were considered to be related to exposure to the test substance.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The findings noted were corneal opacity and persistent pupillary membrane. The incidence or distribution of these findings did not distinguish treated animals from controls.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Histopathological findings: neoplastic:
no effects observed
Details on results:
BODY WEIGHT (Table 2 in "Any other information on results incl. tables")
After 4 weeks of treatment, body weight and body weight gain were considerably reduced in both genders of all treated groups, related to dose, attaining statistical significance in both genders of groups 3 and 4, and in males of group 2 for body weight gain.
After 4 weeks of recovery, body weight was significantly lower in males of group 4 compared to control (group 1). During recovery, body weight gain was considerably higher in both genders of group 4.
For details, see in Table 2.

FOOD CONSUMPTION
During the 4-week treatment period, food consumption was non-significantly reduced in both genders of groups 2 to 4 compared to control (group 1). Relative food consumption was slightly and non-significantly reduced in both genders of groups 2 and 3 and increased in both genders of group 4 compared to control (group 1).
During the recovery period, food consumption and relative food were non-significantly reduced in males of group 4 and increased in females of group 4.

HAEMATOLOGY (Table 3 in "Any other information on results incl. tables")
After 4 weeks of treatment, several haematological parameters were significantly altered in treated animals of both sexes. Overall, treated males displayed significant decreases in erythrocyte count (RBC), haemoglobin (HB), haematocrit (HCT), neutrophil (Neut) count, and in prothrombin time (PT), as well as an increase in reticulocyte (RETI). Treated females showed similar haematological impairments: significant decreases in RBC, HB, HCT, and Neut count, as well as a significant increase in RETI, lymphocyte (Lymph) and monocyte (Mono) counts.
After 4 weeks of recovery, some haematological parameters remained significantly altered; others appeared in treated animals of both sexes. Males of group 4 showed significant decreases in HB, HCT, HDW, total leukocyte count (WBC), basophil (Baso) count, Lymph count, and Mono count. While females of group 4 only displayed a significant decrease in HB.

CLINICAL CHEMISTRY (Table 4 in "Any other information on results incl. tables")
After 4 weeks of treatment, several biochemistry parameters were significantly altered in treated animals of both sexes. Overall, treated males displayed significant increases in glucose, lactate dehydrogenase activity (LDH), chloride, calcium, globulin, as well as significant decreases in urea, total cholesterol, aspartate aminotransferase activity (ASAT), creatinine kinase activity (CK), inorganic phosphorus, and an albumin decrease in group 3 followed by an increase in group 4. Treated females showed the following haematological impairments: significant increases in glucose, triglycerides, alkaline phosphatase (ALP), calcium, inorganic phosphorus, protein, and globulin, as well as significant decreases in urea, creatinine, total cholesterol, CK, sodium, chloride, albumin, and albumin/globulin ratio.
After 4 weeks of recovery, some biochemistry parameters remained significantly altered. Males of group 4 showed only a significant increase in inorganic phosphorus. While females of group 4 displayed significant decreases in calcium, protein, and albumin levels, and a significant elevation in inorganic phosphorus.

In addition, serum α-GST levels were found to be non-significantly decreased in males and females of all treated groups when compared to controls. Regardless of the gender, mean constitutive Inhibin B levels were increased in animals treated for 4 weeks with 0.4 and 0.8 ppm test substance (i.e., groups 3 and 4). After a recovery period of 4 weeks, Inhibin B levels from animals treated with 0.8 ppm test substance returned to control values. The clear decrease of Inhibin B concentrations provided further evidence of gonadotoxicity by the test substance at dose levels of 0.4 and 0.8 ppm. Moreover, T3 levels were significantly decreased in males and females of group 4. Whereas a significant increase in T4 levels was observed in males of groups 3 and 4 and in females of all treated groups. There was no measurement of T3 and T4 levels after the 4-week recovery period.

URINALYSIS
The relative density significantly decreased in males of group 2. Osmolality significantly decreased in males and females of group 2.

ORGAN WEIGHTS (Table 5 in "Remarks on results including tables and figures")
After 4 weeks of exposure, the weight of various organs was significantly altered in treated animals of both sexes. When expressed as organ/body weight ratio, the weight of thymus, adrenals, epididymis, and testes significantly decreased in treated males, while the weight of brain, heart, lungs, liver, kidneys, adrenals, and spleen significantly increased in the same treated groups. Overall, treated females displayed moderate but significant decreases in thymus and ovaries weights and slight but significant increases in brain, liver, and spleen weights (i.e., when expressed as organ/body weight ratio).
After 4 weeks of recovery, some organ weights parameters remained significantly altered. When expressed as organ/body weight ratio, epididymis and testes weights were still significantly decreased and, as seen during the treatment period, the weight of brain, lungs, thymus, and adrenals significantly increased in males of group 4 at the end of the recovery period. The only significant effect seen in females of group 4 was the remaining increase in liver weight (i.e., liver/body weight ratio).

GROSS PATHOLOGY (Table 6 in "Remarks on results including tables and figures")
After 4 weeks of exposure, all males of group 4 and almost all males from group 3 showed a size reduction of testes, epididymis, prostate and seminal vesicles. Two males of group 2 showed seminal vesicles with a reduced size. In all females of group 4, thymus was reduced in size. In groups 3 and 3, a single female demonstrated enlarged liver and ovaries with a reduced size. In one female of group 4, the pituitary gland was thickened.
After 4 weeks of recovery, males of group 4 still showed a size reduction of testes, epididymis, prostate and seminal vesicles. Further, thymus and lymph node discoloration, as well as spleen constriction and skin alopecia/sore were observed in one male of group 4. Two females of group 4 displayed a thickened pituitary gland and alopecia.

HISTOPATHOLOGY: NON-NEOPLASTIC (Table 6 in "Remarks on results including tables and figures")
After 4 weeks of exposure, a minimal centrilobular hepatocellular hypertrophy seen in 2 females of group 2 and a diffuse hepatocellular hypertrophy noted in 2 males of group 2 and in all males and females of groups 3 and 4. This was considered to be possibly adaptive. The hypertrophy seen in males and females of group 4 was associated with minimal to slight midzonal hepatocellular fatty change.Enhanced alveolar histiocytosis occurred in lungs of males and females from group 4.
The pituitary gland of one female from group 4 showed a minimal diffuse hypertrophy of pars distalis.
In addition, diffuse tubular atrophy of testes was seen in 4 males of group 3 and all males of group 4. Atrophy of epididymis occurred in 4 males of groups 3 and 4. An atrophy of seminal vesicles was seen in the majority of all males from all treated groups. An atrophy of prostate was observed in all males of groups 3 and 4. A reduction of the number of corpora lutea in ovaries was described in 1 female of group 3 and 3 females of group 4.
Adrenal cortices presented a minimal diffuse hypertrophy in 2/5 animals of group 3 and in the majority of males and females from group 4. Minimal to slight atrophy of thymus occurred in all males and females of group 4. Both findings were considered to be possibly consequent to stress and/or decreased body weights.
After the 4-week recovery period, the findings in lung, adrenal glands (males only), and male reproductive organs were still present, but mostly in a decreased incidence and/or severity. All other microscopic findings noted in various organs and in all groups examined were considered to be incidental in nature since their morphology, severity, and incidence did not distinguish treated rats from controls.


ENZYMATIC ASSAY
In the additional in vitro analysis, constitutive rat 5’-deiodinase activity in the absence of the test item was found to be 2.578% deiodination/ng protein/min. The test substance clearly inhibited 5’-deiodinase activity in isolated rat liver microsomes, in a concentration-dependent manner. A half maximal inhibitory concentration (IC50) of ca. 5 µM could be calculated for the test item, whereas with 4 µM PTU about 50% of the activity was inhibited.
Human liver microsomes showed ca. 5 times lower constitutive 5’-deiodinase activity than rat liver microsomes (i.e., 0.348% deiodination/ng protein/min). Due to the low activity in human microsomes and the concomitantly variations of individual measurements, a calculation of IC50 could not be performed. However, only a slight inhibition of the deiodinase activity by the test item could be estimated.
Therefore, the test substance was suggested to be an inhibitor of 5’-deiodinase activity in rat liver microsomes whereas in human liver microsomes only a slight inhibition of 5’-deiodinase activity was observed.
Dose descriptor:
NOAEC
Effect level:
< 0.2 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on clinical signs; decreased body weight; food consumption; haematology and clinical chemistry; gross pathology; organ weights; histopathology
Dose descriptor:
NOAEC
Effect level:
< 0.004 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
LOAEC
Effect level:
0.2 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on clinical signs; decreased body weight; food consumption; haematology and clinical chemistry; gross pathology; organ weights; histopathology
Dose descriptor:
LOAEC
Effect level:
ca. 0.004 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified

Table 2: Body weight and body weight gain after 4 weeks of treatment and 4 weeks of recovery

Gender

After 4 weeks of treatment

After 4 weeks of recovery

Males

Females

Males

Females

Group

1

2

3

4

1

2

3

4

1

4

1

4

Body weight relative to control

-

-5.8%

-15.5%*

-30.7%*

-

+0.3%

-13.5%*

-14.2%*

-

-12.9%*

-

-2.5%

Body weight gain

+30.9%

+21.6%*

+3.9%*

-12.1%*

+12.0%

+9.3%

-2.0%*

-6.8%*

+15.3%

+45.1%*

+4.9%

+24.8%*

* = statistically significant

 

Table 3: Summary of significant haematology data after 4 weeks of treatment and 4 weeks of recovery

Gender

After 4 weeks of treatment

After 4 weeks of recovery

Males

Females

Males

Females

Group

1

2

3

4

1

2

3

4

1

4

1

4

RBC (T/L)

8.65

8.17

7.62*

7.58*

8.01

7.45*

7.30*

7.29*

8.95

8.45

8.03

8.01

HB (mmol/L)

9.4

9.2

8.7*

8.2*

9.3

8.8*

8.6*

8.5*

9.7

9.4*

9.5

9.1*

HCT (rel.)

0.42

0.40

0.38*

0.36*

0.41

0.39*

0.39*

0.38*

0.43

0.42*

0.42

0.40

Coagulation - PT (rel.)

0.75

0.70

0.67*

0.72

0.78

0.71*

0.70

0.74

0.80

0.80

0.80

0.79

HDW (mmol/L)

2.10

1.93

1.97

2.11

1.82

1.70

1.77

1.92

2.18

1.75*

1.69

1.71

RETI - Abs (g/L)/Rel.

178/0.21

245*/0.030

267*/0.035*

303*/0.041*

210/0.026

215/0.029

273/0.037*

466*/0.064*

190/0.021

234/0.028

193/0.024

172/0.021

WBC (g/L)

7.62

6.72

6.60

6.10

5.72

4.59

5.38

5.00

7.79

5.37*

4.42

4.41

Neut - Abs (g/L)/Rel.

1.12/0.144

0.98/0.152

0.69*/0.107

0.82/0.134

0.89/0.159

0.62/0.134

0.59/0.108*

0.93/0.183

1.45/0.184

1.04/0.218

0.58/0.129

0.86/0.192

Baso - Abs (g/L)/Rel.

0.03/0.003

0.03/0.004

0.02/0.003

0.02/0.003

0.02/0.003

0.02/0.004

0.02/0.004

0.02/0.003

0.03/0.004

0.01*/0.002

0.02/0.005

0.02/0.004

Lymph - Abs (g/L)/Rel.

6.25/0.822

5.49/0.812

5.68/0.860

5.05/0.828

4.60/0.801

3.83/0.834

4.58/0.851*

3.75/0.749

5.98/0.770

4.11*/0.740

3.65/0.828

3.35/0.762

Mono - Abs (g/L)/Rel.

0.10/0.014

0.10/0.015

0.10/0.015

0.11/0.019

0.09/0.015

0.05/0.011

0.10/0.018

0.20*/0.041

0.18/0.024

0.11*/0.020

0.08/0.018

0.09/0.020

* = statistically significant; rel. = relative

 

Table 4: Summary of significant biochemistry data after 4 weeks of treatment and 4 weeks of recovery

Gender

After 4 weeks of treatment

After 4 weeks of recovery

Males

Females

Males

Females

Group

1

2

3

4

1

2

3

4

1

4

1

4

Glucose (mmol/L)

6.91

7.33

8.99*

6.30

5.91

6.81

8.06*

7.38

6.02

5.52

5.74

5.80

Urea (mmol/L)

6.30

4.90*

5.80

6.33

7.12

6.09

5.82*

6.40

5.83

6.48

8.91

7.35

Creatinine (µmol/L)

24.4

24.9

25.9

26.4

28.7

25.6*

25.7

22.8*

24.8

26.3

34.0

29.7

Cholesterol (mmol/L)

1.49

0.44*

0.40*

1.13

1.59

0.62*

0.97

1.34

1.59

1.74

1.35

1.47

Triglycerides (mmol/L)

0.38

0.79

0.64

0.72

0.32

0.33

0.39

1.29*

0.97

0.46

0.49

0.47

ASAT (U/L)

70.8

61.7

61.3

60.1*

62.3

55.0

51.0

60.0

68.1

74.6

63.6

57.6

LDH (U/L)

130.6

121.4

128.3

212.7*

149.4

87.8

152.6

201.3

122.2

128.0

122.0

129.2

ALP (U/L)

96.7

91.4

88.3

103.8

34.7

41.5

45.3

64.4*

79.6

68.7

23.2

28.3

CK (U/L)

152.7

123.8

99.8*

129.2

126.4

82.4*

90.5

120.9

126.2

141.3

118.9

146.0

Sodium (mmol/L)

143.2

143.1

144.6

144.2

144.6

145.0

144.7

142.9*

142.9

143.9

142.6

142.6

Potassium (mmol/L)

3.58

3.40

3.63

3.56

3.19

3.27

3.69*

3.57

3.46

3.31

2.92

3.06

Chloride (mmol/L)

102.8

103.9

104.6*

103.8

105.1

105.3

105.2

101.5*

102.1

103.1

103.2

104.1

Calcium (mmol/L)

2.61

2.55

2.56

2.72*

2.69

2.69

2.65

2.87*

2.65

2.60

2.72

2.63*

Phosphorus (mmol/L)

2.22

1.97*

1.97*

2.07

1.67

1.53

1.61

1.98*

1.93

2.13*

1.31

1.59*

Protein (g/L)

61.94

62.30

62.24

71.23*

70.38

73.11

68.84

75.27*

66.45

62.68

74.10

68.71*

Albumin (g/L)

39.30

37.75

36.44*

42.35*

48.87

49.43

42.34*

47.21

41.31

38.77

50.57

45.24*

Globulin (g/L)

22.64

24.55*

25.80*

28.88*

21.51

23.68

26.50*

28.05*

25.14

23.92

23.53

23.47

Albumin/globulin ratio

1.74

1.54

1.42

1.47

2.27

2.10

1.60*

1.69*

1.65

1.63

2.17

1.97

Inhibin B (pg/mL)

179

143

54*

89*

179

143

54*

89*

-

184

-

184

T3

108.9

99.0

90.8

57.5*

101.4

110.9

104.9

63.1*

-

-

-

-

T4

6.6

10.2*

9.9*

7.7

3.5

6.6*

7.7*

7.0*

-

-

-

-

* = statistically significant
Conclusions:
Based on the results of this study, a no-observed-adverse-effect-concentration (NOAEC) could not be established for the test substance. Therefore, the NOAEC of the test item was considered to be lower than 0.198 ppm.
Target organs identified in all dose groups of this 4-week rat inhalation study with the test substance were the male reproductive organs (adverse character) and the liver (adaptive nature). Furthermore, adverse effects on the thyroid hormone metabolism were observed in all dose groups.
Executive summary:

The purpose of this 4-week inhalation toxicity study was to evaluate the range of toxicity and to indicate potential target organs (if any) associated with the exposure of the test substance when administered to rats by whole-body inhalation for 6 hours/day for 5 days/week for 4 consecutive weeks. In order to assess the reversibility of any treatment-related findings, satellite groups of animals were maintained for a subsequent period of 4 weeks without treatment.

 

Groups of 5 male and 5 female Wistar rats were exposed to the test substance at target concentrations of 0.2, 0.632, 2 ppm for groups 2 to 4, respectively. The rats of group 1 served as air controls. Additional satellite groups of 5 males and 5 females were kept in groups 1 and 4 to investigate the potential for recovery. Mortality, clinical signs, body weight, food consumption, water consumption, ophthalmoscopic examinations, clinical laboratory investigations, organ weights, macroscopic and microscopic findings were recorded.

 

The mean analytical vapour concentrations of the test substance were 0.198, 0.635 and 1.99 ppm for groups 2 to 4 during phase 1 (exposure days 1 to 12), then 0.198, 0.434 and 0.78 ppm during phase 2 (exposure days 13 to 20). Due to signs of overt toxicity (body weight loss, emaciation), the vapour concentrations for groups 3 and 4 were lowered starting on day 13 of exposure. Temperature, relative humidity and oxygen concentration during exposure were considered to be suitable for this type of study.

 

There were no unscheduled deaths during the course of the study.

 

After 4 weeks of exposure, a pronounced increase in total serum T4 levels was observed in females of all dose groups and in males of groups 3 and 4. Conversely, considerably lower total serum T3 levels were observed in both genders of group 4. The pronounced increase in T4 was considered to lead to an increase in the basal metabolic rate which resulted in reduced body weight development as observed in both genders of all dose groups.

 

In tissues outside the thyroid, particularly in the liver and kidneys, T4 is converted in T3, an active metabolite, by mono-deiodination of the outer phenol ring. It was hypothesised that the substance acted as inhibitor if the (hepatic) de-iodinase leading to an accumulation of T4 and a concomitant decrease in T3. This suggested mode of action was investigated by determination of in vitro conversion of T4/T3 catalysed by rat and/or human liver microsomes in presence of the test substance. The test substance was showed in vitro to inhibit the 5’-deiodinase activity in rat liver microsomes, whereas in human liver microsomes only a slight inhibition of 5’-deiodinase activity was observed.

 

The liver was established as target organ of the test substance by the observation of hepatocellular hypertrophy, reflected by increased liver weight, in both genders of all dose groups although it was considered to be of adaptive nature.

 

Atrophy of testes, epididymis, prostate glands and seminal vesicles in males of groups 3 and 4 correlated with reduced size of those glands. For testes and epididymis, there was a considerable decrease in weight in relation to dose. These findings in male reproductive organs were still present after 4 weeks of recovery and were deemed to be of adverse character. Testicular lesions were confirmed by the significant decrease in Inhibin B levels at the 2 highest doses. Levels returned to normal after the 4 -week recovery period.

In addition, atrophy along with reduced size was also observed in seminal vesicles of males from group 2 and was deemed to be of adverse character.

 

The atrophy in male reproductive organs was considered to be induced by the changes in thyroid hormones via altered endocrine feedback-regulation of TSH/TRH. Hypertrophy of the pituitary gland was observed in one female from group 4.

  

Based on the results of this study, a no-observed-adverse-effect-concentration (NOAEC) could not be established for the test substance. Therefore, the NOAEC of the test item was considered to be lower than 0.198 ppm (0.00448 mg/L).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
4.48 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Good quality study.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The substance induced significant decrease in body weight and food consumption by both the oral and inhalation routes. The higher dose in the 28-day inhalation study caused emaciation and severe body weight loss and test concentrations had to be lowered in the mid- and high-dose groups after 2 weeks of exposure.

Some of the effects were similar for both exposure routes: increased organ weights (liver, kidney, brain, adrenals), or decreased organ weights accompanied by microscopic changes such as atrophy (testes, epididymis, thymus), decreased red blood cells and hemoglobin levels. In addition, in the oral study, the prostate and seminal vesicles weights were found lower in all treated groups, while the weight of lungs, thyroid and hypophysis were increased. White blood cells and lymphocytes were also found decreased.

Histopathological lesions were found in hepatocytes (hypertrophy or swelling, presence of large nucleoli), and could be indicative of metabolic adaptation. Lesions in reproductive organs consisted in atrophy of seminiferous tubules in testes and epididymis, prostate and seminal vesicles in males, decreased corpora lutea in females. Effects on reproductive organs were still present at the end of the recovery period (4 weeks in the inhalation study, and 2 weeks in the oral study). In males, decreased activity of Inhibin B in the 2 higher dose groups of the 28-day inhalation study was consistent with testicular damage.

The liver effects may represent an adaptative response as evidenced by increased liver weight, liver enlargement, hepatocytes hypertrophy or swelling, decreased cholesterol levels and increased total proteins. Most of these liver effects were reversible (except liver weight still higher in the 2 weeks recovery period of the oral study). However the intensity of some of these effects at doses (0.635/0.434 and 1.99/0.78 ppm, i.e. 0.01438/0.00938 and 0.0451/0.0177 mg/L) below the guidance values, and the presence of adverse effects on other organs that could be secondary to the metabolic disturbance, tend to support classification as STOT RE1.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only study available.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
The longest study period was selected. Effects were however consistent with results observed at higher doses in the 5-day dose range finding assay.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
No local effects observed in the 28-day study at up to 2 ppm, as well as in the 5-day dose-range finding study at higher levels, up to 50 ppm.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Repeated dose toxicity: inhalation - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

The liver effects observed by the inhalation and oral routes may represent an adaptative response as evidenced by increased liver weight, liver enlargement, hepatocytes hypertrophy or swelling, decreased cholesterol levels and increased total proteins. Most of the hepatic effects were reversible (except liver weight still higher than controls in the 2 weeks recovery period in the high dose group of the oral study) and thus could usually be considered as non-adverse. However the intensity of some of the effects at doses below the guidance values are considered of concern, and the presence of hormonal dysbalance and adverse effects on other organs and body weight that could be secondary to the metabolic disturbance, tend to support classification as STOT RE1.