3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-ene

Endpoint summary

Description of key information

Short-term oral toxicity (OECD 407, rats): LOAEL = 25 mg/kg bw/day; NOAEL = 5 mg/kg bw/day

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:

key study

Study period:

1 Mar - 24 Aug 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: 28-day Repeated Dose Toxicity Study in Mammalian Species

Version / remarks:

prescribed in "Concerning Testing Methods Relating to the New Chemical Substances" [Notification No. 1121002 of the Pharmaceutical and Food Safety Bureau, MHLW, No. 2 (November 13, 2003) ofthe Manufacturing Industries Bureau, METI & No. 031121002 of the Environmental Health Department, MOE (November 21, 2003)].

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Hino Breeding Center, Shiga, Japan
- Age at study initiation: 5 weeks
- Weight at study initiation: 127.3 – 146.1 g (males) and <111.4 – 130.7 g (females)
- Housing: individual in hanging stainless steel cages with wire-mesh floor. Diet and housing material were autoclaved at 121 °C for 30 min prior to use. Undertrays were changed once a week before grouping, and twice a week after grouping. Feeders, cages and racks were changed once at grouping, and once at termination of the dosing period for the recovery group. Racks and cages were identified by individual cards.
- Diet: MF pelleted diet, ad libitum
- Water: chlorinated water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5 - 24.1
- Humidity (%): 47.9 - 58.6
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

PREPARATION
The test substance was accurately weighed in an agate mortar and mixed with olive oil (including 1.0% (w/v) Tween 80) to prepare the 2.0% (w/v) formulation under a light shielding condition. The lower concentration formulations of the 0.05 and 0.25% (w/v) were prepared by diluting the 2.0% (w/v) formulation with the vehicle. The formulations were stored at the dark and cold place in the test substance preparation room).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Since the hydrolyzability of the test substance was unknown, olive oil (including 1.0% (w/v) Tween 80) was selected as the appropriate vehicle.
- Concentration in vehicle: 0.05, 0.25 and 2.0% (w/v)
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): olive oil (Lot No. 0380HS, Fujimi Pharmaceutical); Tween 80 (Lot No. DPK6694, Wako Pure Chemical Industries)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In the homogeneity analysis, top, middle and bottom layers were taken immediately after preparation, and quantitatively analyzed by gas chromatography after sample pretreatment. The homogeneity of the test substance in the formulations was confirmed.
In the stability test, the formulations for homogeneity samples were stored at the dark and cold place. Then, middle layers of the formulations were taken after 9 days, and quantitatively analyzed by gas chromatography after sample pretreatment. The test substance in the formulations was confirmed to be stable for 8 days.
The concentrations of the 2.0, 0.25 and 0.05% (w/v) formulations were confirmed to be within 100 ± 10% of each nominal concentration at the first preparation of the dosing period.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 days/week

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 (control and high-dose level); 5 (low- and mid-dose level)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A 7-day oral range finding study was performed at 0, 25, 250, 500 and 1,000 mg/kg/day in the same testing facility. Enlargement of the liver and increases in liver weight were noted in the groups given 250 mg/kg or more. Accordingly, the high dose was set at 200 mg/kg/day and lower doses of 25 and 5 mg/kg were set for the present study. Recovery groups were also set for the 200 mg/kg and vehicle control groups.
- Rationale for selecting satellite groups: Recovery groups for the high-dose level and conrol group were used to investigate the reversibility of the effects.
- Post-exposure recovery period in satellite groups: 14 d

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: three times a day during treatment period, twice a day during recovery period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before dosing, once weekly during dosing and recovery periods

BODY WEIGHT: Yes
- Time schedule for examinations: day 1, 3, 8, 12, 17, 21, 26 and 28

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 28 during the dosing period, day 14 during recovery period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- Parameters checked: white blood cell count (WBC), differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cells), red blood cell count (RBC), reticulocyte ratio, prothrombin time (PT), activated partial thromboplastin time (APTT), haemoglobin (Hb), haematocrit (Ht), mean corpucular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 28 during the dosing period, day 14 during recovery period
- Animals fasted: Yes
- Parameters checked: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), cholinesterase, γ-Glutamyl transpeptidase (γ-GTP), total cholesterol, triglyceride, glucose, total protein, albumin, A/G ratio, blood urea nitrogen, creatinine, total bilirubin, calcium, , inorganic phosphorus, sodium, potassium, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: day 28 during the dosing period, day 14 during the recovery group
- Metabolism cages used for collection of urine: Yes
- Paramters checked: urine volume, color, turbidity, urine specific gravity, pH, protein, glucose, occult blood, urinary sediments

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organs checked: liver, kidneys, adrenal glands, testes, epididymides, thymus, spleen, brain, heart, ovaries

HISTOPATHOLOGY: Yes
- Organs checked: trachea, lung, incisors, stomach, intestine (duodenum to rectum, with Peyer`s patches), liver, heart, kidneys, urinary bladder, testes, epididymides, prostate, seminal vesicles, ovaries, uterus, vagina, brain (cerebrum, cerebellum and pons), spinal cord, sciatic nerve, bone marrow (femur), axillar and mesenteric lymph nodes, spleen, thymus, pituitary gland, thyroid (with parathyroids), adrenals, eye balls

Statistics:

Data regarding body weights (excluding those at the time of necropsy), food intakes, hematological examinations, blood chemical examinations, urine volume, urine specific gravity, organ weights, grip strength and locomotor activity counts were analyzed by using the Bartlett's test for homogeneity of variance. If the variances were homogeneous at a significance level of 5%, one way analysis of variance was performed. If there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment group was analyzed by the Dunnett's test. If the variances were not homogeneous, the Kruskal-Wallis's test was used. If there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment group was analyzed by the nonparametric Dunnett's test.

The frequencies of defecation (number of feces) and urination (number of pools) were analyzed by using the Kruskal-Wallis's test. If there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment group was analyzed by the nonparametric Dunnett's test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

MALES
Administration period:
200 mg/kg bw: soft stool (3/10), salivation (9/10)
25 mg/kg bw: salivation (4/5)
5 mg/kg bw: salivation (5/5)
control: salivation (10/10)
Recovery period:
200 mg/kg bw: mottled teeth (2/5)

FEMALES
Administration period:
200 mg/kg bw: salivation (10/10), soft stool (4/10), diarrhea (1/10), loss of hair (forelimb, 1/10)
25 mg/kg bw: salivation (3/5), soft stool (1/5), diarrhea (1/5)
5 mg/kg bw: salivation (1/5), soft stool (1/5), diarrhea (1/5), loss of hair (forelimb, 1/5)
control: salivation (3/10), scab formation (right shoulder, 1/10), loss of hair (forelimb, 1/10), loss of hair (left forelimb, 1/10), loss of hair (right shoulder, 1/10)
Recovery period:
200 mg/kg bw: loss of hair (forelimb, 1/10)
control: loss of hair (forelimb, 1/10)

Salivation was considered to be a physiological response rather than a sign of systemic toxicity as it was observed after dosing and no change related to the neural system was observed in the histopathological examinations, detailed clinical observations of the sensorimotor function. Soft stool or diarrhea were single occurrence. As no histopathological change was observed, these effects were considered to be incidental. Loss of the hair in the forelimbs occurred only in one animal each of the control group, low- and high-dose group and was therefore not considered to be treatment-related.

The summary of clinical signs is shown in Table 1 under "any further information on results incl. tables".

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

FEMALES
5 mg/kg bw/day: significant increase in WBC compared with the control group, since this increase was not observed in the mid- and high-dose group it was not considered to be treatment-related.

MALES
200 mg/kg bw/day recovery group: significant increase of neutrophils and significant decrease of lymphocytes and large unstained cells was noted compared with the recovery control group. This effect was not noted in the other male high-dose group therefore the effect was not considered to be treatment-related.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

MALES
200 mg/kg/day: Significant increase in absolute (30%) and relative (36%) liver weight, significant decrease in absolute heart weight (12%), and absolute (31%) and relative (27%) spleen weights were observed compared with the control group.
200 mg/kg/day recovery: Relative kidney weight (10%) and absolute weight of epididymis (10%) were significantly increased compared with the recovery control group.
25 mg/kg bw/day: Significant increased relative liver weight (13%) compared with the control group was determined.

FEMALES
200 mg/kg bw/day: Relative (8%) kidney weight and relative (15%) liver weight were significantly increased compared with the control group.
25 mg/kg bw/day: Absolute (21%) and relative (14%) liver weights were significantly increased compared with the control group.

The increases of absolute and relative liver weights in the mid- and high-dose groups, respectively, were considered to be treatment-related with toxicological relevance, as these effects occurred in both sexes and were dose-dependent. Furthermore, changes in liver, such as centrilobular lipid droplets in hepatocytes and microgranuloma, were observed in the male high-dose group, in the male high-dose recovery group and occasionally in the female mid- and high-dose group after histopathological examination, please refer to "Histopathological findings, non-neoplastic". In addition to the influence on liver weights and the changes observed after histopathological examination, an enlargement of the liver in all high-dose males compared with the control group was noted after magroscopic examination, please refer to "Gross pathological findings".

The summary of absolute and relative organ weights is shown in Table 2 under "any further information on results incl. tables".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

MALES
At termination of dosing period:
200 mg/kg bw/day: enlargment of the liver (5/5)
25 mg/kg bw/day: cyst of the pituitary gland (1/5)
5 mg/kg bw/day: whitish region on the capsule of the spleen (1/5)
control: sparsed fur on the skin (1/5)

At termination of recovery period:
200 mg/kg bw/day: mottled teeth (3/5)

FEMALES
At termination of dosing period:
200 mg/kg bw/day: elevated region of the mucosa in the forestomach (1/5)
control: scab formation of the skin (1/5)

At termination of recovery period:
200 mg/kg bw/day: mottled teeth (1/5), loss of hair (forelimb, 1/5)
control: loss of hair (forelimb, 1/5)

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

MALES
At termination of dosing period:
200 mg/kg bw/day: centrilobular lipid droplets in the hepatocytes of the liver (3/5 slight and 2/5 moderate), slight periportal hypertrophy of the hepatocytes of the liver (1/5), slight periportal prominent nucleoli of the hepatocytes of the liver (1/5), slight microgranuloma of the liver (4/5), slight degeneration of the spermatocytes of the testis (1/5), slight germ cell debris in the lumen of the epididymis (1/5)
25 mg/kg bw/day: slight centrilobular lipid droplets in the hepatocytes of the liver (1/5), moderate microgranuloma of the liver (1/5), moderate cyst formation in the pars intermedia of the pituitary gland (1/5)
5 mg/kg bw/day: slight capsultitis of the spleen (1/5)
control: slight focal necrosis in the Peyer`s patches of the jejunum (1/5)

At termination of recovery period:
200 mg/kg bw/day: centrilobular lipid droplets in the hepatocytes of the liver (3/5 slight and 1/5 moderate), slight microgranuloma of the liver (3/5)
control: slight mineralization in the medulla of the kidneys (1/5), moderate inhibited spermiation and deep retention of the spermatids of the testis (1/5)

FEMALES
At termination of dosing period:
200 mg/kg bw/day: slight centrilobular lipid droplets in the hepatocytes of the liver (1/5), slight microgranuloma of the liver (2/5), slight lymphocyte infiltration in the submucosal layer of the forestomach (1/5), slight edema in the submucosal layer of the glandular stomach (1/5), slight mineralization in the cortico-medullary junction of the kidney (1/5)
25 mg/kg bw/day: slight peliportal lipid droplets in the hepatocytes of the liver (1/5), slight microgranuloma of the liver (1/5)
control: slight focal inflammation of the rectum (1/5), slight ulcer of the skin (1/5), slight microgranuloma of the liver (1/5)

At termination of recovery period:
control: slight microgranuloma of the liver (1/5)

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects observed at the lowest dose level

Key result

Dose descriptor:

LOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Table 1 Summary of clinical signs

Signs	Dosing Period						Recovery Period
	VC	VC (Recovery)	5 mg/kg bw/d	25 mg/kg bw/d	200 mg/kg bw/d	200 mg/kg bw/d (Recovery)	VC	200 mg/kg bw/d
MALE	5 animals / group
No abnormalities detected		2		1		1	5	3
Salivation	5	3	5	4	5	4
Soft stool					2	1
Mottled teeth								2
Loss of hair (ventral neck)
FEMALE	5 animals / group
No abnormalities detected	1	4	2	2			4	4
Salivation	3		1	3	5	5
Soft stool			1	1	2	2
Diarrhea			1	1		1
Loss of hair (right shoulder)	1
Loss of hair (forelimb)		1				1	1	1
Loss of hair (left forelimb)			1
Loss of hair (right shoulder)		1

VC = Vehicle control

Table 2 Summary of absolute (g) and relative (g/100g)) organ weights in males

Exp. Group [mg/kg/day]	Number of animals	Liver (g)	Liver (g/100g)	Heart (g)	Spleen (g)	Spleen (g/100g)
VC	5	10.07 ± 1.02	3.09 ± 0.24	1.14 ± 0.10	0.70 ± 0.14	0.22 ± 0.05
5	5	11.08 ± 1.51	3.26 ± 0.12	1.16 ± 0.05	0.60 ± 0.07	0.18 ± 0.02
25	5	11.39 ± 1.69	3.50 ** ± 0.19	1.08 ± 0.10	0.58 ± 0.06	0.18 ± 0.02
200	5	13.03 ** ± 1.08	4.22 ** ± 0.13	1.00 * ± 0.07	0.48 ** ± 0.07	0.16 * ± 0.02
VC (Recovery)	5	10.85 ± 2.73	2.83 ± 0.34	1.23 ± 0.16	0.65 ± 0.12	0.17 ± 0.01
200 (Recovery)	5	11.17 ± 1.07	3.00 ± 0.15	1.24 ± 0.12	0.56 ± 0.04	0.15 ± 0.02

VC = Vehicle control

* Significantyl different from vehicle control at P<0.05.

** Significantyl different from vehicle control at P<0.01.

Conclusions:

Based on the effects on the increased liver weights, the enlarged liver size and histopathological findings in the liver at 25 mg/kg bw/day, the substance is classified as STOT-RE 1, H372, oral, liver.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

25 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Subacute toxicity

The registered substance was tested in a repeated dose oral toxicity study similar to OECD guideline 407 and in compliance with GLP (Key, 2012). Male and female Wistar rats were treated via gavage with test substance at concentrations of 5, 25 and 200 mg/kg bw/day, respectively. 5 animals per sex and dose were used at the low- and mid-dose level and 10 animals were used at the high-dose level and as control. The selected dose levels were based on the results of a range-finding study, in which adverse effects were observed in rats administered 250, 500 and 1,000 mg/kg bw/day for up to 7 days. The control group received the vehicle olive oil. In the high dose group salivation was observed in 9/10 males and 10/10 females after dosing. This was considered to be a physiological response rather than a sign of systemic toxicity as it was observed after dosing and no change related to the neural system was observed in the histopathological examinations and detailed clinical observations of the sensorimotor function. Further clinical signs such as soft stool, diarrhea and loss of the hair in the forelimbs were considered to be incidental findings. No treatment-related findings were noted in functional observations, food consumption and body weight gain, in haematological findings and in the urinalysis. Mean relative liver weight was increased up to 13% and 36% at 25 and 200 mg/kg bw/day in males, respectively, and up to 15% and 14% at 25 and 200 mg/kg bw/day in females, respectively. The increase of absolute and relative liver weights in the mid- and high-dose groups were considered to be treatment-related with toxicological relevance in both mid- and high-dose groups, as these effects occurred in both sexes and were dose-dependent. Furthermore, the changes in liver were supported histopathologically by centrilobular lipid droplets in hepatocytes and microgranuloma in males and females. These effects were observed in the male high-dose group, in the male high-dose recovery group and occasionally in the female mid- and high-dose group after histopathological examination. In addition, gross pathological examination revealed an enlargement of the liver in all high-dosed males. Based on the results of the conducted study, a No Observed Adverse Effect Level (NOAEL) was determined to be 5 mg/kg bw/day and the Low Observed Adverse Effect Level (LOAEL) was determined to be 25 mg/kg bw/day for male rats.

Justification for classification or non-classification

The available data on oral repeated dose toxicity with the test substance meet the criteria for classification according to Regulation (EC) No 1272/2008 and therefore the test substance will be classified as STOT RE Cat. 1 (H372) with liver as target organ.