Tetraethylammonium heptadecafluorooctanesulphonate

Endpoint summary

Description of key information

A 90 day oral feed study of PFOS in rats and several not assignable (secondary literature) subacute and subchronic gavage or capsule sudies in monkeys were conducted. Additional a 104-week dietary chronic toxicity and carcinogenicity study with perfluorooctane sulfonic acid potassium salt (PFOS) in rats is as secondary literature available. Based on the liver toxicity, the no-observed-adverse-effect level (NOAEL) for PFOS is considered to be 0.5 ppm (0.5  ppm corresponds with 0.017-0.057 mg/kg body weight/day ) in male rats and 2 ppm (2.0 ppm corresponds with 0.095-0.213 mg/kg bw/day) in female rats. 
In the rat subchronic study CD rats, 5/sex/group, were administered dietary levels of 0, 30, 100, 300, 1000 or 3000 ppm PFOS (FC-95) for 90 days. The dietary levels were equivalent to doses of 0, 2, 6, 18, 60 and 200 mg/kg/day. A NOAEL was not identified in this study.
In the first ninety-day subacute rhesus monkey toxicity study doses of 0, 10, 30, 100 or 300 mg/kg/day PFOS were applied by gavage. all animals died. In the second ninety-day subacute rhesus monkey toxicity study doses of 0, 0.5, 1.5 or 4.5 mg/kg/day PFOS (FC-95) in distilled water by gavage
were administerd. A NOAEL was not found, the LOAEL 0.5 mg/kg/day. 
Additional, PFOS (potassium perfluoroctane sulfonate) was administered to cynomolgus monkeys by oral capsule at doses of 0 (6 monkeys per sex), 0.03 (4 monkeys per sex), 0.15 (6 monkeys per sex), or 0.75 mg/kg/day (6 monkeys per sex) for 26 weeks. The NOAEL = 0.15 mg/kg bw/d. PFOS is toxic to cynamolgous monkeys at 0.75 mg/kg/day causing death, alterations in total cholesterol, and effecting liver weight and causing hepatocellular hypertrophy and vacuolation in both treated males and females. In the 28 day capsule dose range finding study no NOAEL was determined.
As secondary literature a 104-Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS) in rats is available.  

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Dose descriptor:

NOAEL

0.15 mg/kg bw/day

Study duration:

subchronic

Species:

monkey

Additional information

A 90 day oral feed study of PFOS in rats all of the rats in the 300, 1000 and 3000 ppm groups died. Death occurred between days 13-25 and days 18-28 for the males and females, respectively, in the 300 ppm group. At 1000 ppm, death occurred between days 8-14, and at 3000 ppm, the rats died between days 7-8 of treatment. The rats in all groups showed signs of toxicity including emaciation, convulsions following handling, hunched back, red material around the eyes, yellow material around the anogenital region, increased sensitivity to external stimuli, reduced activity and moist red material around the mouth or nose. Three males and two females in the 100 ppm group died prior to scheduled sacrifice. Two of the males and the two females died during week 5 and the third male died during week 11 of the study. At study termination, mean body weights were reduced by 16.7% and 16.3% in the male and female groups, respectively. Average food consumption during the entire study period (g/rat/day) was significantly reduced for males and females at 100 ppm. After 30 days of treatment, hematologic values were comparable among the control and 100 ppm groups. Clinical chemistry analyses at one month showed a significant increase in mean glucose in males, blood urea nitrogen values in males and females, and creatinine phosphokinase and alkaline phosphatase values for females. After 90 days of treatment at 100 ppm, the two surviving males had significantly reduced erythrocyte, hemoglobin, hematocrit and leukocyte counts; the three surviving females had significantly reduced hemoglobin and reticulocyte counts, as well as slightly lower erythrocyte, hematocrit and leukocyte counts. Two of the surviving females showed slight to moderate increases in plasma glutamic oxalacetic and pyruvic transaminase activities. Urinalysis results were comparable among treated and control groups at 30 and 90 days. Relative liver weight was significantly increased in the males and absolute and relative liver weights were significantly increased in the females. Relative kidney weights were significantly increased in both sexes.

All rats in the 30 ppm group survived until the end of the study. At study termination, mean body weights were reduced by 8.7 and 8% in the males and females, respectively. Average food consumption during the entire study period (g/rat/day) was significantly reduced for the males at 30 ppm. Hematologic values were comparable among the control and 30 ppm group at 30 and 90 days. One female showed a slightly elevated glucose level and one male showed a slightly increased alkaline phosphatase level at 30 days. At 90 days, one male showed moderate increases in glucose, blood urea nitrogen and y-glutamyl transpeptidase activity. The females had significant increases in absolute and relative liver weights. The males had significant decreases in absolute and relative adrenal weights, absolute thyroid/parathyroid weight and absolute pituitary weight. The biological significance of the changes in male organ weights is unclear since similar changes were not noted in higher dose groups. At necropsy, treatment related gross lesions were present in all treated groups and included varying degrees of discoloration and/or enlargement of the liver and discoloration of the glandular mucosa of the stomach. Histologic examination also showed lesions in all treated groups. Centrilobular to midzonal cytoplasmic hypertrophy of hepatocytes and focal necrosis was observed in the liver; the incidence and relative severity were greater in the males. In addition, especially among rats in the 300, 1000 and 3000 ppm groups, treatment related histologic lesions were noted in the primary (thymus, bone marrow) and secondary (spleen, mesenteric lymph nodes) lymphoid organs, stomach, intestines, muscle and skin. In the thymus, this consisted of depletion in the number and size of the lymphoid follicles and in the bone marrow hypocellularity was noted. The spleen was slightly atrophied with a corresponding decrease in the size and number of lymphoid follicles and cells and a similar depletion was noted in the mesenteric lymph nodes. Mucosal hyperkeratosis and/or acanthosis was observed in the forestomach and mucosal hemorrhages were noted in the glandular portion of the stomach. Decreases in the height and thickness of the villi were noted in the small intestine. Atrophy of the skeletal muscle was noted, as well as epidermal hyperkeratosis and/or acanthosis was noted in the skin.

In the 26 -week capsule toxicity study with perfluorooctane sulfonic acid potassium salt (PFOS; T-6295) in cynomolgus monkeys

males weighed 3.3-3.4 kg and females weighed 2.8-2.9 kg at the beginning of the study. At the end of 26 weeks of treatment, males weighed 3.7, 3.8, 3.5, and 3.3 kg for the 0, 0.03, 0.15 and 0.75 mg/kg/day treatment groups respectively. Females weighed 3.1, 3.1, 3.1 and 2.8 kg for the 0, 0.03, 0.15 and 0.75 mg/kg/day treatment groups respectively. The difference between the control and the 0.75 mg/kg/day female treatment groups was statistically significant. At the end of the recovery period, differences in weight between the control and treated animals were no longer obvious.

Two males from the 0.75 mg/kg/day group did not survive to the scheduled sacrifice. One animal died after dosing on Day 155 (Week 23). Clinical signs noted in this animal included: constricted pupils, pale gums, few, mucoid, liquid and black-colored feces, low food consumption, hypoactivity, labored respiration, dehydration, and recumbent position. In addition, the animal was cold to the touch. An enlarged liver was detected by palpation. Cause of death was determined to be pulmonary necrosis with severe acute

inflammation. On day 179, the second male was sacrificed in a moribund condition. Clinical signs noted included low food consumption, excessive salivation, labored respiration, hypoactivity and ataxia. The cause of death was not determined.

Males and females in the 0.75 mg/kg/day dose-group had lower total cholesterol and males and females in the 0.15 and 0.75 mg/kg/day groups had lower high density lipoprotein cholesterol during treatment. The effect on total cholesterol worsened with time. By day 182, mean total cholesterol for males and females in the high dose group were 68% and 49% lower, respectively, that than levels in the control animals. The effect on high density lipoprotein was greater than that seen with cholesterol. On day 182, the mean high density lipoprotein levels were 79% and 62% lower, respectively in males and females from the high dose group than they were in male and female control animals. Males in the high dose group also had lower total bilirubin concentrations and higher serum bile acid concentrations than males in either the control or other treatment groups. The effect on total cholesterol was reversed within 5 weeks of recovery and the effect on high density lipoprotein cholesterol was reversed within 9 weeks of recovery.

Estradiol values were lower in males given 0.75 mg/kg/day on days 62, 91, and 182 but because of variation only the day 182 value was significant. Estrone values were generally higher in the treate females on days 37, 62 and 91 but again because of variation in the data none of these values were significantly different from the controls. Except for males in the 0.15 mg/kg/day group, triiodothyronine

values were significantly lower on days 91 and 182 in males and females given 0.15 and 0.75 mg/kg/day.

There were other instances in which hormone values in treated groups were different from those of controls but these differences were not consistent over time or between sexes, were not clearly dose-related and did not appear to be related to the administration of the test material. Apparent differences in the sexual maturity of both males and females used in the study complicates the interpretation of the hormone data.

At terminal sacrifice, females in the 0.75 mg/kg/day dose-group had increased absolute liver weight, liver to-body weight percentages, and liver-to-brain weight ratios. In males, liver-to body weight percentages were increased in the high-dose group compared to the controls. “Mottled” livers were observed in two high-dose males and in one high-dose female. Of the two males not surviving until the scheduled terminal sacrifice, one had a “mottled” and large liver. Three of 4 high-dose males (including those that did not survive to scheduled sacrifice) had centrilobular or diffuse hepatocellular hypertrophy that was also observed in all high-dose females. Centrilobular or diffuse hepatocellular vacuolation occurred in 2 of 4 females and 2 of 4 males in the high-dose group.

No PFOS related lesions were observed either macroscopically or microscopically at recovery sacrifice indicating that the effects seen at terminal sacrifice may be reversible.

Although low levels of PFOS were often detected in the sera and liver of the control animals, these levels were significantly lower than those found in the low dose test animals. PFOS levels in the sera of test animals increased with dose during treatment from 21.0 ± 1.57 and 20.4 ± 2.71 µg/ml in the Group 4 males and females respectively at the end of Week 1 to 194 ± 8.94 and 160 ± 23.1 µg/ml in males and females respectively in Group 4 at the end of Week 27. During recovery, PFOS levels in serum samples decreased over time until they reached 41.1 ± 25.9 µg/ml in males and 41.4 ± 1.15 µg/ml in females from Group 4 at 79 weeks post-treatment. Control values were < LOQ (the limit of quantitation) at Week 4 in both males and females and 0.0215 ± 0.00296 and 0.0243 ± 0.00355 µg/ml in males and females respectively at the end of Week 79. The serum values for selected weeks of treatment and recovery are shown in the table below. There were no significant differences between PFOS levels in the sera of treated males and females.

Concerning the 104 -week dietary chronic toxicity and carcinogenicity study with perfluorooctane sulfonic acid potassium salt (PFOS) in rats there was a significant increased trend in survival that occurred in the males that was due to significant increases in survival in mid-high (5.0 ppm) and high-dose (20.0 ppm) groups as compared to that of the control group. None of the other treated groups in the males revealed any significant differences in survival. No significant trend was noted in survival in females. There was a significant decrease in survival in the mid-dose (2.0 ppm) group and not in the mid-high (5.0 ppm) and high-dose (20.0 ppm) groups as compared to that of the control.

Males given 20 ppm had significantly lower mean body weights compared to animals in the control group during weeks 9 through 37. Females given 20 ppm had significantly lower body weights compared to animals in the control group during weeks 3 through 101. At week 105, mean body weights of the surviving males and females were not significantly different from the controls. Food consumption for males and females was similar in all treated groups compared to animals given the control material except for the high-dose females which had statistically significantly lower food consumption during weeks 2 through 44.

At the week 14 and week 53 interim sacrifice, absolute and relative liver weights were significantly increased in the males given 20 ppm. In females given 20 ppm, only the liver-to-body weight percentage was significantly increased. Treatment-related histomorphologic changes were seen in the liver in the males given 5 or 20 ppm and in the females given 20 ppm. The changes consisted of hypertrophy of hepatocytes in centrilobular areas in males and females, and midzonal to centrilobular hepatocytic

vacuolation. The incidence and severity of the changes tended to be greater in the males. Dietary administration of PFOS for approximately 53 weeks was associated with mildly to moderately lower cholesterol for males and females fed 20 ppm; and mildly higher alanine aminotransferase for males fed 20 ppm. In the unscheduled sacrifices between Weeks 54 and 105, animals given 20 ppm had increased hepatocellular centrilobular hypertrophy, eosinophilic hepatocytic granules, and centrilobular hepatocytic

pigment were noted. Increased hepatocellular centrilobular hypertrophy was seen in animals given 5 ppm.

At the terminal sacrifice, the livers of animals given 5 or 20 ppm exhibited a slight increase in macroscopic findings, including enlarged, mottled, diffuse darkened, or focally lightened. Hepatotoxicity, characterized by significant increases (P<0.05) in centrilobular hypertrophy, centrilobular eosinophilic hepatocytic granules, centrilobular hepatocytic pigment, or centrilobular hepatocytic vacuolation was noted in male and/or female rats given 5 or 20 ppm. A significant increase (P<0.05) in hepatocellular centrilobular

hypertrophy was also observed in mid-dose (2 ppm) male rats. Significant increases in the incidence of cystic hepatocellular degeneration was found in all the male treated groups (0.5, 2, 5, or 20 ppm); however, this lesion is believe to be due to old age of the animals and is not considered to be treatment-related.

Based on the pathological findings in the liver, the no-observed-adverse-effect level (NOAEL) for PFOS is considered to be 0.5 ppm in male rats and 2 ppm in female rats; the low observed-adverse-effect level (LOAEL) is 2 ppm in male rats and 5 ppm in female rats.

There was no effect on hepatic palmitoyl-CoA oxidase activity. There were also no statistically significant increases in cell proliferation as measured by proliferative cell nuclear antigen (PCNA) at weeks 4 and 14, or by bromodeoxyuridine (BrdU) at week 53.

For neoplastic effects, a significant positive trend (P=0.0276) was noted in the incidences of hepatocellular adenoma in male rats. This was due to a significant increase (P<0.05) in the high-dose group (11.7%, 7/60) over the control (0%, 0/60). A significantly increased incidence (P<0.05) was observed for thyroid follicular cell adenoma in the high-dose recovery group (23.1%, 9/39) when compared to the control group (5%, 3/60). There was also a slight increase in the combined thyroid follicular cell adenoma and

carcinoma in the high-dose recovery group (25.6%, 10/39) as compared to that of the control group (10%, 6/60); the increase did not reach statistical significance relative to the control but did reach statistical significance relative to the high-dose group (8.5%, 5/59).

In the females, significant positive trends were observed in the incidences of hepatocellular adenoma (P=0.0153) and combined hepatocellular adenoma and carcinoma (P=0.0057). These cases were due to significant increases in the high-dose group (8.3%, 5/60, and 10%, 6/60) as compared to the control (0%, 0/60). A significant increase (P=0.0471) for combined thyroid follicular cell adenoma and carcinoma was observed in the mid-high (5.0 ppm) group (6%, 3/50) as compared to the control group (0%, 0/60). Except for the high-dose group (which showed a slight decrease in incidences of mammary fibroadenoma/adenoma and combined mammary fibroadenoma and carcinoma), increases in mammary tumors were observed in all treatment groups when compared to the controls. Significant increases (P<0.05) in mammary fibroadenoma/adenoma (60%, 30/50) and combined mammary fibroadenoma/ adenoma and carcinoma (72%, 36/50) were observed in the low–dose (0.5 ppm) group as compared to the respective controls (38.3%, 23/60 and 48.3%, 29/60). The mid-dose (2.0 ppm) group also exhibited a statistically significant (P<0.05) increase (64.6%, 31/48) in the incidence of combined mammary fibroadenoma/adenoma/carcinoma over the control group (43.8%, 29/60). Increases in mammary tumors in the mid-high (5 ppm) dose group did not reach statistical significance relative to the control.

Read-across justification

- Group: The perfluorooctane sulfonate anion (PFOS) does not have a specific CAS number. The acid and salts have the following CAS numbers: acid (1763-23-1), ammonium (NH4 +) salt (29081-56-9), potassium (K+) salt (2795-39-3), tetraethylammonium (C2H5)4N+) salt (56773-42-3).

For the registration of tetraethylammonium perfluorooctanesulfonate (CAS 56773-42-3) the data on perfluorooctane sulfonate anion (PFOS) as well as the respective salts were taken into consideration and a read across approach was used due to the following reasons:

- Justification: REACH regulation EC 1907/2006 and ECHA guidance document R.6 state that substances whose physico-chemical, toxicological and ecotoxicological properties are likely to be similar, or follow a regular pattern as a result of structural similarity, may be considered as a group / category of substances. Properties of PFOS are mainly determined by the length of the fluorinated tail and not by the nature of the functional group. PFOS and its salts are dissociated in aqueous media, substituted amines and the corresponding sulfonyl fluorid are hydrolysed in aqueous media to PFOS. Therefore these compounds are members of a large family of perfluoroalkyl sulfonate substances. According to OECD, 2002, (ENV/JM/RD(2002)17/FINAL) “perfluorinated compounds represent a very unique chemistry”, at least substances with equal numbers of perfluorinated carbon atoms and functional groups.

Thus, in accordance with OECD, 2002, grouping and read-across based on the length of the perfluorinated carbon structure with a sulfonate moiety is in principle possible. The criterion of structural similarity is fulfilled and all substances may be regarded as group with respect to chemical behaviour. This judgement is in line with and confirmed by the existing classification of perfluoroalkyl sulfonates in Europe: according Annex VI to Regulation (EC) No 1272/2008 for classification and labeling several perfluoroalkyl sulfonates are considered as a group/category and therefore identical hazard class/category codes, and hazard statement codes applies. The following substances/CAS number are mentioned explicitly in the regulation: perfluorooctane sulfonic acid/1763-23-1, potassium perfluorooctanesulfonate/ 2795-39-3, diethanolamine perfluorooctane sulfonate/70225-14-8, ammonium perfluorooctane sulfonate/29081-56-9, lithium perfluoroocane sulfonate/29457-72-5.

This approach also apply to tetraethylammonium perfluorooctanesulfonate, as the evaluation of the toxicological data of tetraethylammonium perfluorooctanesulfonate substantiate the membership of the substance to the group and the same hazard class and statement codes are valid.

Therefore based on the available data as well as on earlier judgements by e.g. OECD and within the EU classification a read across approach is justified.

Read-across justification

- Group: The perfluorooctane sulfonate anion (PFOS) does not have a specific CAS number. The acid and salts have the following CAS numbers: acid (1763-23-1), ammonium (NH4 +) salt (29081-56-9), potassium (K+) salt (2795-39-3), tetraethylammonium (C2H5)4N+) salt (56773-42-3).

For the registration of tetraethylammonium perfluorooctanesulfonate (CAS 56773-42-3) the data on perfluorooctane sulfonate anion (PFOS) as well as the respective salts were taken into consideration and a read across approach was used due to the following reasons:

- Justification: REACH regulation EC 1907/2006 and ECHA guidance document R.6 state that substances whose physico-chemical, toxicological and ecotoxicological properties are likely to be similar, or follow a regular pattern as a result of structural similarity, may be considered as a group / category of substances. Properties of PFOS are mainly determined by the length of the fluorinated tail and not by the nature of the functional group. PFOS and its salts are dissociated in aqueous media, substituted amines and the corresponding sulfonyl fluorid are hydrolysed in aqueous media to PFOS. Therefore these compounds are members of a large family of perfluoroalkyl sulfonate substances. According to OECD, 2002, (ENV/JM/RD(2002)17/FINAL) “perfluorinated compounds represent a very unique chemistry”, at least substances with equal numbers of perfluorinated carbon atoms and functional groups.

Thus, in accordance with OECD, 2002, grouping and read-across based on the length of the perfluorinated carbon structure with a sulfonate moiety is in principle possible. The criterion of structural similarity is fulfilled and all substances may be regarded as group with respect to chemical behaviour. This judgement is in line with and confirmed by the existing classification of perfluoroalkyl sulfonates in Europe: according Annex VI to Regulation (EC) No 1272/2008 for classification and labeling several perfluoroalkyl sulfonates are considered as a group/category and therefore identical hazard class/category codes, and hazard statement codes applies. The following substances/CAS number are mentioned explicitly in the regulation: perfluorooctane sulfonic acid/1763-23-1, potassium perfluorooctanesulfonate/ 2795-39-3, diethanolamine perfluorooctane sulfonate/70225-14-8, ammonium perfluorooctane sulfonate/29081-56-9, lithium perfluoroocane sulfonate/29457-72-5.

This approach also apply to tetraethylammonium perfluorooctanesulfonate, as the evaluation of the toxicological data of tetraethylammonium perfluorooctanesulfonate substantiate the membership of the substance to the group and the same hazard class and statement codes are valid.

Therefore based on the available data as well as on earlier judgements by e.g. OECD and within the EU classification a read across approach is justified.

Justification for classification or non-classification

Based on the result of the 90 day oral feed study in rats, the 26 weeks capsule study in monkeys and the 104 -week dietary chronic toxicity and carcinogenicity study with perfluorooctane sulfonic acid potassium salt as classification as R48 (GHS: STOT RE 1) is justified.