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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
screening
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
02AUG2012-11DEC2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
This screening study did not include all the parameters of the standard guideline. Only selected organs and parameters were examined. Non-GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Treatment was similar to a 28-day study, but only a few parameters were investigated, focused on thyroid and sexual hormones as well as reproductive organs (organ weights and macroscopic examination)
GLP compliance:
no
Remarks:
Preliminary mechanistic study not conducted under GLP, but in a GLP facility
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1,2,2,3,3,4,4-octafluoro-1,4-diiodobutane
EC Number:
206-788-4
EC Name:
1,1,2,2,3,3,4,4-octafluoro-1,4-diiodobutane
Cas Number:
375-50-8
Molecular formula:
C4F8I2
IUPAC Name:
1,1,2,2,3,3,4,4-octafluoro-1,4-diiodobutane
Test material form:
other: liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1,4-diiodoperfluorobutane, DA02 C4 puro, batch No. 0629SP
- Expiration date of the lot/batch: 31 December 2020
- appearance: violet clear liquid
- Purity: 98.2%
- Purity test date: 31/07/2012

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient condition, protected from light
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: no data

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: 27-29 days old
- Weight at study initiation: 75-99 g
- Fasting period before study: No
- Housing: 5 of one sex to a cage, in clear polysulfone solid bottomed cages measuring 59x38.5x20 cm (Code 1354 G, Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
- Diet : ad libitum
- Water : ad libitum)
- Acclimation period: approximately 2 weeks was allowed before the start of treatment

DETAILS OF FOOD AND WATER QUALITY:
- laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy)
- Records of analyses of water and diet are kept on file at the test facility

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2°C
- Humidity (%): 55% +/- 15%
- Air changes (per hr): 15 - 20 ACH
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: From: 22-AUG-2012 To: 15-OCT-2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
suspension
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test substance was suspended in the vehicle (corn oil). The formulation was prepared daily at the concentrations of 0.1, 0.3, 1 and 3 mg/mL. Concentrations were calculated and expressed in terms of test item as supplied. Animals were administered 10 ml/kg bw.

VEHICLE
- Justification for use and choice of vehicle: no details provided
- Concentration in vehicle: see above
- Amount of vehicle (if gavage): 10 ml/kg bw
- Lot/batch no. (if required): no data
- Purity: no data
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
1 mg/kg bw/day (nominal)
Dose / conc.:
3 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 per sex and per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on information from preliminary studies.
- Rationale for animal assignment: Randomisation. Animals at the extremes of the weight distribution were excluded to leave the required number of animals. The rats were allocated to
the 5 groups by computerised stratified randomisation to give approximately equal initial group mean body weights
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Not examined
DETAILED CLINICAL OBSERVATIONS: All clinical signs were recorded for individual animals. Observation before commencement of treatment, then once daily.

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 30-mg/kg groups

FOOD CONSUMPTION AND COMPOUND INTAKE: Not examined
FOOD EFFICIENCY: Not examined
WATER CONSUMPTION AND COMPOUND INTAKE: Not examined
OPHTHALMOSCOPIC EXAMINATION: Not examined
HAEMATOLOGY: Not examined
CLINICAL CHEMISTRY: Not examined
URINALYSIS: Not examined
NEUROBEHAVIOURAL EXAMINATION: Not examined
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Epididymides
Liver
Ovary
Pituitary gland
Testis
Thyroid gland

HISTOPATHOLOGY: No
Other examinations:
ORGAN WEIGHTS (absolute and relative):
Epididymides
Liver
Ovary
Pituitary gland
Testis
Thyroid gland
Statistics:
standard deviations were calculated
statistical analysis: for continuous variables, the significance of the differences amongst group means was assessed by Dunnett's test if group variances were homogeneous ; Modified t test if group group variances were inhomogeneous

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
High dose animals showed marked clinical signs, such as emaciated appearance, hunched posture and piloerection. Decreased muscle tone was observed in 2 females of the high dose group. No relevant clinical signs were observed in the other groups including controls.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One high dose group female was sacrificed for human reasons. Findings at macroscopic examination showed mis-dosing.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the administration period, males and females treated with the high dose levels (30 mg/kg bw/day) showed mean body weight significantly lower than control group, especially in males, and starting from day 8. No remarkable differences in body weight were noted in the other treatment groups.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In this study, clinical chemistry was limited to selected hormones: Thyroid hormones (T3, T4), TSH, LF were analysed. See in "Details on results"
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological 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"
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
ORGAN WEIGHT (Tables 1 & 2 in "Any other information on results incl. Tables")
After 28 days of treatment, the weight of various organs was significantly altered in treated animals of the high dose group (30 mg/kg bw/day):
- significantly reduced absolute and relative epididymides and testes weights in males.
- significantly increased relative (+66%) liver weight in males, while females showed statistically significant increase in both absolute (+24%) and relative (+66%) liver weight.
- no change was observed in the absolute thyroid and pituitary weights in both sexes. Increase in relative pituitary weight was only statistically significant in males, while increase in relative thyroid weight was statistically significant in both sexes.
- in females, a lower absolute (-15%) ovaries weight and a slightly higher relative (+14%) ovaries weight were observed, although not statistically significant.

GROSS PATHOLOGY (Table 3 in "Any other information on results incl. Tables")
The main macroscopic observations are summarized in table 3. The main changes consisted in reduced organ size for epididymides, prostate, and seminal vesicles in all males of group doses at 30 mg/kg bw/day, and 2 out of 5 males at 10 mg/kg bw/day. All males also had reduced testes size at 30 mg/kg bw/day.
Enlarged and/or pale colour were observed in the liver of treated males, while swollen liver was seen in treated males, in a single female dosed at 10 mg/kg bw/day, and in one control male.
Swollen pituitary was noted in a couple of treated males and few treated females and in a single control female. One female doses at 30 mg/kg bw/day showed yellow staining in the cutis of the urogenital region.
The remaining changes could be considered incidental in origin or an expression of spontaneous pathology, normally seen in this species and laboratory.

CLINICAL CHEMISTRY (see figure under "attached background material")
Circulating Thyroid Hormones (free T3, T4), Thyroid Stimulating Hormone (TSH) and Luteinizing Hormone (LH) were analysed in serum at different time points during the treatment period to detect any change in hormone homeostasis.
Increased circulating levels of FT4 and TSH were observed in treated groups, especially in animals (males and females) receiving 10 and 30 mg/kg/day, starting from Day 6 of study. On the contrary, FT3 levels appeared reduced in males of the treated groups compared to the control group.
FSH appeared to increase in males of the high dose group 30 mg/kg/day. Variability between animals and time points did not allow observing a clear dose-dependent pattern. The corresponding data on females were not clear.
LH values were below the limit of quantitation (0.1 ng/ml) in almost all animals for all time points.

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day (nominal)
System:
endocrine system
Treatment related:
yes
Dose response relationship:
yes

Any other information on results incl. tables

Table 1: Organ weights after 28-day treatment - Males

Gender

Males

Dose (mg/kg bw/day)

0

1

3

10

30

LIVER

-      Absolute (g)

-      Relative wt. (%)

 

16.794

4.087

 

15.289

3.900

 

19.335

4.480

 

16.814

4.437

 

17.664

6.543**

TESTIS

-      Absolute (g)

-      Relative wt. (%)

 

3.2250

0.7889

 

3.4098

0.8711

 

3.2976

0.7641

 

2.9086

0.7765

 

0.9112**

0.3376**

EPIDIDYMIS

-      Absolute (g)

-      Relative wt. (%)

 

1.2074

0.2994

 

1.2550

0.3203

 

1.1540

0.2674

 

0.9776

0.2604

 

0.3002**

0.1115**

THYROID

-      Absolute (g)

-      Relative wt. (%)

 

0.0232

0.0057

 

0.234

0.0060

 

0.0246

0.0057

 

0.0250

0.0066

 

0.0234

0.0088**

PITUITARY

-      Absolute (g)

-      Relative wt. (%)

 

0.0118

0.0029

 

0.0122

0.0031

 

0.0106

0.0025

 

0.0110

0.0029

 

0.0136

0.0052**

*p<0.05 and ** p< 0.01

 

Table 2: Organ weights after 28-day treatment - Females

Gender

Females

Dose (mg/kg bw/day)

0

1

3

10

30

LIVER

-      Absolute (g)

-      Relative wt. (%)

 

11.112

4.169

 

10.453

40.020

 

10.688

4.230

 

12.330

4.831

 

13.786*

6.946**

OVARIES

-      Absolute (g)

-      Relative wt. (%)

 

0.1154

0.0433

 

0.1304

0.0500

 

0.1238

0.0493 

 

0.1094

0.0429

 

0.0978

0.0494

THYROID

-      Absolute (g)

-      Relative wt. (%)

 

0.0238

0.0089

 

0.0220

0.0085

 

0.0228

0.0091

 

0.0220

0.0086

 

0.0240

0.0122**

PITUITARY

-      Absolute (g)

-      Relative wt. (%)

 

0.0146

0.0055

 

0.0124

0.0048

 

0.0132

0.0053

 

0.0136

0.0053

 

0.0120

0.0061

*p<0.05 and ** p< 0.01

Table 3: Main macroscopic findings 28-day treatment - Males

Gender

 

 

Males

 

 

Dose (mg/kg bw/day)

0

1

3

10

30

LIVER

-      Abnormal area(s)

-      Abnormal colour

-      Abnormal shape

-      Abnormal size

 

0

0

1

0

 

0

1

0

0

 

0

0

4

0

 0

1

1

0

 

0

2

2

1

EPIDIDYMIDES

- abnormal size

 

0

 

0

 

0

 

2

 

5

PROSTATE

- abnormal size

 

0

 

0

 

0

 

2

 

5

SEMINAL VESICLES

- abnormal size

 

0

 

0

 

0

 

2

 

5

TESTIS

- abnormal size

 

0

 

0

 

0

 

0

 

5

 

Table 4: Main macroscopic findings 28-day treatment - Females

Gender

Females

Dose (mg/kg bw/day)

0

1

3

10

30

LIVER

-      Abnormal area(s)

-      Abnormal shape

 

0

0

 

0

0

 

0

0

 

0

1

 

1

0

OVARY

- abnormal size

 

0

 

3

 

2

 

1

 

0

PITUITARY

- abnormal shape

 

1

 

1

 

2

 

2

 

1

Applicant's summary and conclusion

Executive summary:

The purpose of this 28-day oral toxicity screening study was to investigate the effects of the test substance on the thyroid and sexual hormones serum levels.

Groups of 5 male and 5 female Sprague-Dawley rats were exposed to the test substance at dose levels of 0 (vehicle control), 1, 3, 10, and 30 mg/kg body weight (bw)/day. Mortality, clinical signs, body weight, organ weights, macroscopic findings were recorded.

After 28 days of treatment, terminal body weight was statistically reduced in both sexes of the high dose groups. Significantly reduced absolute and relative epididymides and testes weights were found in males receiving the highest dose of 30 mg/kg/day. Significantly increased liver weight was found in high dose males (relative weight) and females (absolute and relative weight). Relative thyroid and/or pituitary weights to body weight were significantly increased in the high dose males, while both sexes showed increased relative thyroid weights.

In females, a lower absolute ovaries weight and a slightly higher relative ovaries weight were observed, although not statistically significant.

 

The main macroscopic changes consisted in reduced organ size for epididymides, prostate, and seminal vesicles in all males of group doses at 30 mg/kg bw/day, and 2 out of 5 males at 10 mg/kg bw/day. All males also had reduced testes size at 30 mg/kg bw/day. Enlarged and/or pale colour were observed in the liver of treated males, while swollen liver was seen in treated males, in a single female dosed at 10 mg/kg bw/day, and in one control male. Swollen pituitary was noted in a couple of treated males and few treated females and in a single control female.

Circulating Thyroid Hormones (free T3, T4), Thyroid Stimulating Hormone (TSH) and Luteinizing Hormone (LH) were analysed in serum at different time points during the treatment period to detect any change in hormone homeostasis. Increased circulating levels of FT4 and TSH were observed in treated groups, especially in males and females receiving 10 and 30 mg/kg/day, starting from Day 6 of study. On the contrary, FT3 levels appeared reduced in males of the treated groups compared to the control group. FSH appeared to increase in males of the high dose group 30 mg/kg/day. Variability between animals and time points did not allow observing a clear dose-dependent pattern. The corresponding data on females were not clear. LH values were below the limit of quantitation in almost all animals for all time points.