Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoate

Additional toxicological data

Administrative data

Endpoint:

additional toxicological information

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Cross-reference

Reason / purpose for cross-reference:

other: reevaluation of liver sections

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Remarks:

The study was conducted according to guideline in effect at time of study conduct.

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

other: Crl:CD1(ICR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 7 weeks old.
- Weight at study initiation:
Mean Body Weights of Male Mice by group: Control - 31.8 g, 0.1 mg/kg - 32.1 g, 0.5 mg/kg - 32.2 g, 5 mg/kg - 32.2 g
Mean Body Weights of Female Mice by group: Control - 25.9 g, 0.1 mg/kg - 25.8 g, 0.5 mg/kg - 25.8 g, 5 mg/kg - 26.3 g
- Housing: Animals were housed individually in solid bottom caging with bedding and nestlets as enrichment. Each cage rack contained only animals of one sex.
- Diet: PMI® Nutritional International, LLC. Certified Rodent LabDiet® 5002, ad libitum
- Water: tap water, ad libitum.
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26ºC
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): approximate 12 hour light/dark cycle

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF TEST SUBSTANCE FORMULATION:

- Solvent used: deionized water.
- Preparation frequency: weekly
- Preparation details: The test substance was formulated in deionized water. Dose formulations were prepared weekly with a correction for the sponsor-reported purity of 84%, and stored protected from light until used.
- Adjusted for purity: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Method of Analysis: HPLC

Concentration verification (% of Target))
Conducted on all dose levels: yes
Results:
Week 0: ± 8.2% of Nominal
Week 7: ± 13.2% of Nominal
Week 12: ± 13.2% of Nominal


Stability (% of Time Zero)
Conducted on dose levels: yes
Results:
Week 0: 8-Day Room Temperature Storage: mean of all dose levels 108% of Nominal
Week 0: 15-Day Room Temperature Storage: mean of all dose levels 107% of Nominal

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0.1, 0.5, and 5 mg/kg
Basis:
other: nominal by gavage

No. of animals per sex per dose:

All dose groups consisted of 25 animals/sex, for which the first 10 animals/sex were used in the toxicity evaluations. The remaining 15 animals/sex were used for evaluation of plasma concentration of the test substance (5/sex/dose/timepoint).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: According to the results of a 28-day study, test substance administered orally (gavage) to Crl:CD1(ICR) mice at dosage levels of 0.1, 3 and 30 mg/kg/day for 28 consecutive days was well tolerated. Most test substance-related effects were consistent with a peroxisome proliferator (PPARα agonist) and included increased beta-oxidation in the liver, increased liver weights, hepatocellular hypertrophy, and changes in serum lipids and proteins. Other changes included increased body weights, minimal decreases in red cell mass parameters and increased adrenal weights and adrenal cortical hypertrophy. These effects were reversible following a 4-week recovery period. Adverse effects were noted in the liver of males at 3 mg/kg/day and above and females at 30 mg/kg/day. These consisted of single cell necrosis of hepatocytes and correlative increases in liver enzymes. These effects were also reversible following the 4-week recovery period.
The no-observed-adverse-effect level (NOAEL) for oral (gavage) administration of test substance to Crl:CD1(ICR) mice for 28 consecutive days was 0.1 mg/kg/day for males and 3 mg/kg/day in females.

Based on the results of the 28-day study, doses of 0, 0.1, 0.5, and 5 mg/kg/day were selected for this study. The 5 mg/kg/day dose was expected to produce effects on clinical chemistry and liver biochemical parameters, organ weight, and/or histopathology in males and females. The 0.5 mg/kg/day dose was thought to possibly produce liver and clinical chemistry changes in males but could be a no-observed-adverse-effect level (NOAEL) in females. The 0.1 mg/kg/day dose was expected to be a NOAEL in both males and females.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were performed daily and detailed physical examinations were performed weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY: Yes
- The amount of food consumed by each animal over each weighing interval was determined by weighing each feeder at the beginning and end of the interval and subtracting the final weight from the initial weight. From these measurements, mean daily food consumption over the interval was determined. From the food consumption and body weight data, the mean daily food efficiency was calculated.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to administration of test substance and at week 12

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on test days 96 and 97 for males and females, respectively
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: no
- How many animals: All animals
- Parameters checked in Table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on test days 96 and 97 for males and females, respectively
- Animals fasted: no
- How many animals: All animals
- Parameters checked in Table No. 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 13

OTHER: PLASMA CONCENTRATION EVALUATION
- How many animals: 5/sex/exposure level/ time point
- Time schedule for examinations: 0-, 28- and 95-day time points.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 3)
HISTOPATHOLOGY: Yes (see Table 3)

A complete necropsy was conducted on all animals. Animals were euthanized by carbon dioxide inhalation and exsanguinated. Microscopic examination was performed on all tissues from all animals in the control and high-dose groups euthanized at the scheduled primary necropsy. All tissues collected from control and high-dose (5 mg/kg/day) mice at the terminal sacrifice (test days 96 and 97), and tissues collected from early decedents were processed to slides and evaluated microscopically. Potential target organs (liver in males and females; kidneys in males only) and gross observations (as recorded at necropsy) were examined microscopically for all animals designated for the subchronic toxicity evaluation.

Statistics:

See Table 4 in section directly below

Clinical signs:

no effects observed

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:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no test substance-related deaths. One control female mouse was found dead on test day 16. One male mouse (0.5 mg/kg/day; test day 3) and 3 female mice (2 at 0.5 mg/kg/day on test day 6 and 1 at 5 mg/kg/day on test day 20) were euthanized due to clinical signs attributed to misdosing. These animals displayed clinical signs related to injury from the gavage misdosing (swollen neck and/or shoulder, abnormal breathing, cold, dehydrated, and/or lethargic; abnormal gait) prior to death.
One male in the 0.1 mg/kg/day group (201) exhibited aggressiveness and convulsions, each on one day, and hyperreactivity over multiple days, especially over the second half of the dosing period. These signs were not considered to be test substance related as they only occurred in one male mouse in the low dose group and were not associated with any correlative pathology findings.
One female in the 5 mg/kg/day group (451) exhibited intermittent signs of hyperactivity, hyperreactivity, and running in circles. Although this female was in the high dose group, these signs were not attributed to test substance exposure as they only occurred in one mouse, they were not seen everyday, the observations were separated by hours and days when the mouse exhibited no abnormal signs, the signs were often observed prior to dosing (when test substance blood levels would have been expected to be at the lowest level) and they were not associated with any correlative pathology findings.
All other clinical observations were typical of those seen in mice of this age and were not considered to be test substance related.

BODY WEIGHT AND WEIGHT GAIN
No adverse, test substance-related effects on mean body weight or mean body weight gain were observed in any male or female group.
Statistically significant increases in mean final body weight (test day 91) and overall body weight gain (test days 0-91) were observed in the male 5 mg/kg/day group, relative to control. Mean final body weight and overall body weight gain were 108% and 136% of control, respectively. Mean body weight in this group was higher than control on all test days (variable statistically significance). Body weight gain was significantly higher than control over 2 weekly intervals (test days 49-56 and 84-91). No statistically significant differences in mean body weight or body weight gain were observed in any other male test group on any test day or over any test week. The difference in body weight and body weight gain in the high dose males was attributed primarily to increased liver weight, and was not considered adverse.
Mean final body weight and overall body weight gain in the 5 mg/kg/day female group were 101% and 98% of control, respectively (neither statistically significant). Mean body weight gain in the female 0.5 mg/kg/day group was significantly higher than control over test days 7-14 and significantly lower than control over test days 28-35, but these differences were considered spurious as overall body weight gain in this group was comparable to control. No other statistically significant differences in body weight or body weight gain were observed in any groups on any test day or over any test week.

FOOD CONSUMPTION AND FOOD EFFICIENCY
No adverse, test substance-related effects on mean food consumption or food efficiency were observed in any male or female group.
Statistically significant increases in mean overall (test days 0-91) food consumption and food efficiency were observed in the male 5 mg/kg/day group, relative to control. Mean overall food consumption and food efficiency were 111% and 127% of control, respectively. Mean weekly food consumption in this group was higher than control over all weeks except test days 0-7 (statistically significant over most intervals). Mean weekly food efficiency was higher than control over the first 6 weekly intervals, but none of the differences was statistically significant. Weekly food efficiency over the rest of the study was variable, with higher and lower mean values (variable statistical significance). The higher food efficiency is likely due to increased body weight due to enlarged livers in this group. No statistically significant differences in mean food consumption or food efficiency were observed in any other male test group on any test day or over any test week.
Mean overall food consumption and food efficiency in the 5 mg/kg/day female group were 102% and 100% of control, respectively (neither statistically significant). No statistically significant differences in food consumption were observed over any weekly interval in this group. Mean food efficiency in the female 0.5 mg/kg/day group was significantly higher than control over test days 7-14 and significantly lower than control over test days 28-35. These differences were correlated with differences in body weight gain over those intervals and were considered spurious as overall food efficiency in this group was comparable to control. No other statistically significant differences in food consumption or food efficiency were observed in any groups on any test day or over any test week.

OPHTHALMOSCOPIC EXAMINATION
No ophthalmological signs were observed in any mouse in any group.

HAEMATOLOGY
There were no adverse or treatment-related changes in group mean hematology parameters at test day 96 (males) or 97 (females). The following statistically significant changes were not considered to be test substance related or adverse.
• Mean corpuscular hemoglobin (MCHC) was minimally decreased in male mice dosed with 5 mg/kg/day (97% of control). MCHC is a calculated hematology parameter using values for hemoglobin, red blood cell and mean corpuscular volume ((hemoglobin/[red blood cell * mean corpuscular volume])* 100). There were no statistically significant decreases in red cell mass parameters (red blood cell, hemoglobin, hematocrit), mean corpuscular volume or changes in red cell morphology. Therefore, minimally decreased MCHC was considered to be spurious.

• Platelet count (PLT) was minimally increased in male mice dosed with 0.5 or 5 mg/kg/day (127% and 126% of control, respectively). Changes in platelet count did not show a dose response relationship despite the 10-fold increase in dose. There were no clinical signs or pathology or clinical pathology changes consistent with biological processes that might be associated with thrombocytosis (e.g., acute or chronic inflammation). In addition, higher platelet counts were not seen in a previous 28 day gavage study(1) with the same test substance. Therefore, the higher platelet counts in the 0.5 and 5.0 mg/kg/day male groups were considered to be unrelated to treatment and non-adverse.

• Absolute monocyte count (AMON) was minimally decreased in female mice dosed with 0.1 mg/kg (25% of control). This decrease was not dose related, as a similar change was not seen in the higher dose groups. Therefore the lower AMON in the 0.1 mg/kg/day female group was not considered to be test substance related or adverse.

CLINICAL CHEMISTRY
See Table 5.
Test substance-related increases in a number of liver-related clinical chemistry parameters were present in male and female mice administered 5 mg/kg/day. Increases were mild to severe, were consistently more severe in males compared to females, and included increases in aspartate aminotransferase (AST), alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH), alkaline phosphatase (ALKP) and total bile acids (TBA). Changes in these parameters were consistent with hepatocellular damage and/or cholestasis, correlated microscopically with hepatocellular single cell necrosis in male (10/10) and female (1/10) mice at this dose, and thus were considered to be adverse effects.
There were no statistically significant changes in liver enzyme parameters in male or female mice administered 0.5 or 0.1 mg/kg/day.

There were no other adverse changes in group mean clinical chemistry parameters in male or female mice. The following statistically significant changes in mean clinical chemistry parameters were not adverse or not related to exposure to the test substance:
• Total protein (TP) and albumin (ALB) were minimally increased in male mice dosed with 5 mg/kg (110% and 114% of control, respectively). ALB was also minimally increased in female mice at the same dose (104% of control). These changes were considered to be treatment-related due to the consistency of change among individual animals. However, minimally increased total protein and albumin have no toxicologic significance; therefore, these changes were considered to be non-adverse.
• Cholesterol (CHOL) was mildly decreased in male mice dosed with 5 mg/kg (74% of control). There are no known adverse effects associated with minimal decreases in cholesterol. Therefore, these changes were considered test substance related but non-adverse.
• Bilirubin (BILI) was minimally decreased in female mice dosed with 5 mg/kg. However, this statistical difference was not considered treatment related or indicative of an adverse effect, The magnitude of difference in BILI was small (86% of control), the individual BILI values in all 5 mg/kg/day females except one were within the concurrent control range, and no changes in BILI were present in male mice, the more sensitive gender. In addition, the direction of change in BILI in 5 mg/kg/day females–decreased rather than increased–is not considered adverse. Therefore, these changes were not considered to be treatment related or adverse.
• Potassium (K) was decreased in male and female mice dosed with 5 mg/kg (87% and 91% of control, respectively). Decreased K typically occurs when there is a shift of K from extra cellular fluid to intracellular fluid (e.g., in metabolic alkalosis), a decreased dietary intake of K, or an increased loss of K via kidneys (e.g., polyuria), alimentary tract (e.g., diarrhea) or skin (e.g., sweating). In the present study, the relationship of this finding to test substance administration is uncertain. However, there were no clinical signs suggestive of hypokalemia and no test substance-related alterations in sodium (Na). Therefore, the minimal change in K was not considered to be adverse.
• Decreased bilirubin (BILI) in male mice dosed with 0.5 mg/kg (76% of control) was not dose related and was thus considered to be a spurious finding.
• Chloride (CL) was slightly higher (102% of control) in male mice dosed with 5 mg/kg. Based on the minimal nature of the change and lack of any correlative findings, this change was considered to be unrelated to treatment and non-adverse.
In conclusion, under the conditions of this study, adverse changes in liver-related parameters (AST, ALT, SDH, ALKP, TBA) were present in male and female mice dosed with 5 mg/kg/day. No adverse changes in clinical pathology parameters were present in male or female mice dosed with 0.5 or 0.1 mg/kg/day.

NEUROBEHAVIOUR
Under the conditions of the study, the test substance had no effect on neurobehavioral parameters in either males or females.

ORGAN WEIGHTS
See Table 6
A test substance related increase in mean liver weight parameters was observed in mice exposed to ≥ 0.5 mg/kg/day in males and 5 mg/kg/day in females.
In the 5 mg/kg/day males, mean absolute and mean relative (% brain weight and % body weight) liver weights were increased to 263%, 242%, and 230% of control, respectively. These increases were statistically significant. In the 0.5 mg/kg/day males, mean absolute and mean relative (% brain weight and % body weight) liver weights were also increased (not statistically significant) to 112%, 113%, and 111% of control, respectively. In 5 mg/kg/day females, mean absolute and mean relative (% brain weight and % body weight) liver weights were increased (statistically significant) to 169% , 167% and 169% of control, respectively.
Increased liver weight parameters were considered test substance related in males given ≥ 0.5 mg/kg/day and in females given 5 mg/kg/day. The increase in liver weight parameters in both sexes correlated with a treatment-related increase in enlarged liver and microscopic hepatic changes.
Mean relative (to brain) weight of kidneys was increased (statistically significant) in male mice given 5 mg/kg/day of test substance as compared to controls. Although minimal renal tubular hypertrophy was present in this group, the change in kidney weight relative to brain weight was not associated with changes in mean absolute or relative (% body weight) kidney weights. Therefore, the significance of the kidney weight change relative to test substance administration is uncertain.
Mean weights of brain and epididymides relative to body weight were lower, and mean weight of heart relative to brain weight was higher in male mice given 5 mg/kg/day of test substance as compared to controls (all statistically significant). These changes occurred without correlative changes in other weight parameters for these organs or with microscopic findings. Thus, these organ weight changes were not considered to be test substance related.
Mean spleen weight relative to brain and body weight were decreased relative to controls in the 0.5 and 5 mg/kg/day female mice respectively. These changes did not occur in a dose-related manner, were not associated with changes in mean absolute spleen weight, and were not associated with test substance-related microscopic changes in the spleen. In addition, no spleen weight changes occurred in male mice in any dose group. Therefore, the changes in mean spleen weight relative to brain and body weight in females were considered spurious and unrelated to treatment.
In females, mean absolute and mean relative (% brain and % body weight) ovarian weights were increased to 203%, 206%, and 208% of control, respectively. These ovarian weight changes did not occur in dose related manner. Ovaries of 3 female mice (357, 359 and 360) in the 0.5 mg/kg/day exposure group had cysts and their ovarian weights were outside the range of individual values in control or high dose animals. Therefore, the increase in mean ovarian weights in the 0.5 mg/kg/day group is due to the influence of 3 female mice having ovarian cysts. Ovarian cysts in this exposure group were considered as background lesions and no dose response for this lesion was observed. Therefore, the ovarian weight changes in 0.5 mg/kg/day female mice were considered spurious and unrelated to test substance administration.


GROSS PATHOLOGY
See Table 7
At the terminal sacrifice, enlarged and/ or discolored livers were observed in 4/10 and 9/10 male mice exposed to 0.5 mg/kg/day and 5 mg/kg/day of test substance respectively. In the 5 mg/kg/day group 3/10 female mice had enlarged livers. These gross changes were considered test substance related. Liver enlargement and discoloration correlated with test substance related increases in liver weights and microscopic hepatocellular hypertrophy and all other gross observations were consistent with normal background lesions in mice of this age and strain.

HISTOPATHOLOGY: NON-NEOPLASTIC
See Table 8
1. Liver
Test substance related and adverse microscopic findings were present in the liver of male and female mice administered 5 mg/kg/day of the test substance. At 0.5 mg/kg/day, test substancerelated microscopic changes were limited to male mice which had minimal hepatocellular hypertrophy, without evidence of liver cell injury. Therefore, this change was considered to be test substance related but not adverse. There were no test substance-related changes in the liver of male or female mice administered 0.1 mg/kg/day or in female mice administered 0.5 mg/kg/day.
In the 5 mg/kg/day male and female groups, test substance-related hepatocellular hypertrophy was present in all animals. Hypertrophy was graded as mild (grade 2 out of 4) in males and minimal (grade 1 out of 4) or mild in females. The distribution of the hepatocellular hypertrophy was centrilobular when of minimal severity and diffuse when of mild severity. Hypertrophy was morphologically consistent with peroxisome proliferation and was characterized by increase in the size of hepatocytes due to increased amount of finely granular eosinophilic cytoplasm and enlarged nuclei with occasional binucleated cells. Additional liver changes in the 5 mg/kg/day group occurred most consistently in males and included increased numbers of mitotic figures (males only), increased pigment (likely lipofuchsin) in Kupffer cells, and single cell hepatocellular necrosis. The latter change was characterized by isolated eosinophilic bodies with occasional pyknotic nuclear fragments and unaccompanied by inflammation, and thus was consistent with apoptosis. Hepatic lesions correlated with increased absolute and relative liver weight and increased total bile acid and liver enzyme levels (AST, ALT, SDH, ALP). Minimal bile duct hyperplasia was present in the liver of one male mouse in the 5 mg/kg/day group. Since similar changes were not seen in any other treated mice, the relationship of this finding to test substance administration is uncertain.
In the 0.5 mg/kg/day groups, liver changes were limited to minimal hepatocellular hypertrophy in males only. Hypertrophy was not associated with microscopic changes or changes in clinical chemistry parameters indicative of liver cell injury. Therefore, liver hypertrophy in the 0.5 mg/kg/day male group was considered test substance related but non-adverse.
There were no other microscopic findings in liver considered to be test substance related. In females, focal necrosis was present in both treated and control mice with slightly increased incidence in the 5 mg/kg/day females (1/10, 0/10, 2/10, 3/10 in control, 0.1, 0.5, and 5 mg/kg/day groups, respectively). Focal hepatic necrosis is a common background lesion in mice, and there was no difference in morphology or severity of this lesion in treated female mice as compared to controls. In addition, test substance-related focal necrosis did not occur in males, the more sensitive gender for liver effects. Therefore, the minimal increase in the incidence of this lesion in high dose females was considered spurious and unrelated to treatment. All other microscopic changes were consistent with background findings common to mice of this strain and age.

2. Kidney
Test substance related changes in the kidney were limited to minimal tubular epithelial hypertrophy in 9/10 male mice given 5 mg/kg/day of the test substance. Hypertrophy was characterized by slightly enlarged epithelial cells containing increased amounts of fine granular eosinophilic cytoplasm. Tubular epithelial hypertrophy was not associated with renal tubular cell degeneration/necrosis. Also there was no change in clinical pathology parameters indicative of renal injury. Therefore, the renal epithelial hypertrophy was considered a non-adverse adaptive response.
All other microscopic observations in treated and control animals were consistent with normal background lesions in mice of this age and strain.

3. Anatomic Pathology Conclusions
Under the conditions of this study, daily gavage administration of 5 mg/kg of test substance resulted in test substance-related and adverse effects in the liver of male and female mice. Incidences and severity of changes were greater in males compared to females. At 0.5 mg/kg/day, non-adverse liver effects were present in males only. No test substance-related changes were present in females given 0.5 mg/kg/day or in males or female given 0.1 mg/kg/day. An increase in kidney weight and minimal renal tubular epithelial cell hypertrophy was observed in 5 mg/kg/day males but was considered a non-adverse, adaptive response. No test substance-related effects on mortality were observed at any dose.


OTHER FINDINGS
Plasma Concentration Evaluation
The test substance concentration in blood was similar on days 0, 28, and 95 in female mice, indicating that steady-state concentrations were achieved on the first day of dosing. This is consistent with a test substance that was cleared rapidly from the blood within one dosing interval. The test substance concentration in blood from male mice was slightly lower on day 0 than on day 28, but the day 28 and day 95 concentrations were similar, indicating steady state had been achieved by day 28. Compared to female mice, male mice took longer to achieve steady-state concentrations in blood. The plasma concentration was linear with dose, implying that absorption was not saturated over the range of doses tested in this study. Test substance was not present in plasma from control animals.

Dose descriptor:

NOAEL

Effect level:

0.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on changes in clinical chemistry and histopathology indicative of liver toxicity in animals dosed with 5 mg/kg/day

Critical effects observed:

not specified

Table 5.  Clinical Chemistry
	Male Mice – Test Day 96				Female Mice – Test Day 97
Dosage (mg/kg):	0	0.1	0.5	5	0	0.1	0.5	5
AST (U/L)	62	67^a	84	128	68	71	69	74
		108%^b	135%	206%		104%	101%	109%
ALT (U/L)	49	62	66	255	36	36	32	51
		127%	135%	520%		100%	89%	142%
SDH (U/L)	26.6	26.0	25.8	108.5	25.3	22.9	23.6	33.5
		98%	97%	408%		118%	111%	243%
ALKP (U/L)	50	55	70	617	65	77	72	158
		110%	140%	1234%		118%	111%	243%
TBA (μmol/L)	1.2	1.2	1.4	11.1	4.3	2.3	2.7	13.2
		100%	117%	925%		53%	63%	307%
a - mean
b - % of control
Bold = statistically significant

Table 6. Test Substance-Related Effects on Mean Absolute and Relative Liver Weights
Dose (mg/kg/day):	0	0.1	0.5	5
Male
Number of Mice:	10	10	10	10
Mean final body weight (grams)	38.7	40.3	38.9	44.3*
Liver
absolute weight (grams)	1.955	2.024	2.186	5.144*
Liver weight/body weight x 100	5.06	5.028	5.618	11.637*
Female
Number of Mice:	10	10	9	9
Mean final body weight (grams)	32.2	32.1	32.6	32.4
Liver
absolute weight (grams)	1.693	1.697	1.745	2.867*
Liver weight/body weight x 100	5.225	5.309	5.337	8.811*
* Statistically significant as compared to control value.
Underlined values were interpreted to be test-substance related increases, as compared to control values.

Table 7. Test Substance-Related Gross Observations in Mice
	Male				Female
Dose (mg/kg/day):	0	0.1	0.5	5	0	0.1	0.5	5
mice/group:	10	10	10	10	10	10	10	10
Liver	(10)	(10)	(10)	(10)	(10)	(10)	(10)	(10)
Large	0	0	1	9	0	0	0	3
Discoloration	0	0	4	5	1	0	0	3
Numbers in parentheses are the number of tissues examined within each group.
Underlined values were interpreted to be test substance-related gross findings.

Table 8. Incidences of Test Substance-Related Microscopic Findings in the Liver of Male and Female Mice
	Male				Female
Dose (mg/kg/day):	0	0.1	0.5	5	0	0.1	0.5	5
mice/group:	10	10	10	10	10	10	10	10
Liver	(10)	(10)	(10)	(10)	(10)	(10)	(10)	(10)
Hepatocellular hypertrophy	0	0	8	10	0	0	0	10
Hepatocellular single cell necrosis	0	0	0	10	0	0	0	1
Mitotic figures	0	0	0	9	0	0	0	0
Pigment increased, Kupffer cells	0	0	0	10	0	0	0	2
Numbers in parentheses are the number of tissues examined within each group.
Underlined values were interpreted to be test substance-related gross findings.

Conclusions:

Male NOAEL = 0.5 mg/kg/day
Female NOAEL = 0.5 mg/kg/day
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).

Executive summary:

The objective of this study was to evaluate the potential subchronic toxicity of the test substance in mice. Four groups of young adult male and female Crl:CD1(ICR) mice (10/sex/group) were dosed by oral gavage for at least 90 days. Mice were dosed with the test substance at doses of 0 (control), 0.1, 0.5, or 5 mg/kg/day of H-28548. The control mice were dosed with deionized water. Body weights, food consumption, and detailed clinical observations were evaluated weekly and acute clinical observations were evaluated daily. Ophthalmology and neurobehavioral (functional observational battery and motor activity) were evaluated prior to study start and near the end of the dosing period. Clinical pathology endpoints (hematology and clinical chemistry parameters) were evaluated at the end of the exposure period. After 96 (males) or 97 (females) days of dosing, the surviving mice designated for subchronic toxicity evaluation were sacrificed and given a gross and microscopic pathological examination. An additional 15 mice/sex/dose were included in each group and were evaluated for plasma concentration of the test substance in samples collected approximately 2 hours after dosing on test days 0, 28, and 95 (5/sex/dose/timepoint). These mice were evaluated for body weight, food consumption, clinical signs, and ophthalmology (pretest only) but did not receive neurobehavioral or clinical or anatomic pathology evaluations.

No test substance-related deaths occurred, and no clinical observations were attributed to exposure to the test substance. One male and 4 female mice died prior to scheduled sacrifice, due to gavage trauma. No adverse, test substance-related effects on body weight, body weight gain, food consumption, or food efficiency were observed. Non-adverse increases in body weight and nutritional parameters were observed in males in the 5 mg/kg/day group, but were attributed primarily to increased liver weights observed in this group. No test substance-related effect on any neurobehavioral parameter was observed in any dose group.  

 

There were no adverse changes in hematology parameters that were attributed to the test substance exposure. Increases in the level of multiple liver enzymes and total bile acids were observed in 5 mg/kg/day males and females and correlated with microscopic liver pathology effects observed at that dose. Minimal or mild increases in total protein and albumin and mildly reduced cholesterol levels were observed in males at 5 mg/kg/day. Minimally increased albumin was observed in females at 5 mg/kg/day. These protein and cholesterol changes were considered treatment related but non-adverse.

 

In 5 mg/kg/day male and female dose groups, increases were observed in the incidence of single cell necrosis, mitotic figures, and/or Kupffer cell pigment. The liver effects at 5 mg/kg/day correlated with clinical chemistry effects and were considered test substance related and adverse. Other test substance-related effects were observed in the livers of 0.5 and 5 mg/kg/day males and 5 mg/kg/day females, including increases in absolute and/or relative liver weight, enlarged and/or discolored livers, and centrilobular hepatocellular hypertrophy. The liver effects observed in 0.5 mg/kg/day males were considered to be non-adverse adaptive responses as they were not correlated with clinical or microscopic pathology evidence of liver toxicity. Increased kidney weights and minimal tubular epithelial hypertrophy were observed in 5 mg/kg/day males but were considered to be non-adverse adaptive responses.

Results of analysis of plasma from multiple time points in females were consistent with a test substance that was cleared rapidly from the blood within one dosing interval, and that reached steady state on the first day of dosing. Compared to female mice, male mice took longer to clear the test substance from the blood, with steady state being achieved by test day 28. The plasma concentration was linear with dose, implying that absorption was not saturated over the range of doses tested in this study. Test substance was not present in plasma from control animals. 

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for the test substance was 0.5 mg/kg/day in males and females, based on changes in clinical chemistry and histopathology indicative of liver toxicity in animals dosed with 5 mg/kg/day. 

 

Applicant's summary and conclusion

Executive summary:

HFPO-DA is an alternative to environmentally persistent long-chain perfluoroalkyl and polyfluoroalkyl substances. Mice exposed to HFPO-DA exhibit liver hypertrophy, elevated peroxisomal enzyme activity, and other apical endpoints consistent with peroxisome proliferators. To investigate the potential role of peroxisome proliferator-activated receptor alpha (PPARα) activation in mice, and other molecular signals potentially related to observed liver changes, RNA sequencing was conducted on paraffin-embedded liver sections from a 90-day subchronic toxicity study of HFPO-DA conducted in mice. Differentially expressed genes were identified for each treatment group, and gene set enrichment analysis was conducted using gene sets that represent biological processes and known canonical pathways. Peroxisome signaling and fatty acid metabolism were among the most significantly enriched gene sets in both sexes at 0.5 and 5 mg/kg HFPO-DA; no pathways were enriched at 0.1 mg/kg. Gene sets specific to the PPARα subtype were significantly enriched. These findings were phenotypically anchored to histopathological changes in the same tissue blocks: hypertrophy, mitoses, and apoptosis. In vitro PPARα transactivation assays indicated that HFPO-DA activates mouse PPARα. These results indicate that the liver changes observed in HFPO-DA-treated mice occur via a mode of action (MOA) involving PPARα, an important finding for human health risk assessment as this MOA has limited relevance to humans.