Fatty acids, sunflower-oil, conjugated, maleated,reation products with diethanolamine, maleated tall-oil fatty acids and triethanolamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 421 (RA to TEA): increased post-implantation loss at 1000mg/kg (BASF 2010)

OECD 443 (RA to DEA): The following NOAELs were obtained (BASF 2018):

- General toxicity F0/F1: 100ppm / 13mg/kg (liver, kidney, hematopoietic system)

- Fertility: F0/F1: 300ppm / 38mg/kg (increased gestation time, prolonged estrous cycle)

- Developmental Toxicity: 300ppm / 38mg/kg (Lower number of implants, reduced pub survival, feminization of male mammary glands, effects on testes and ovaries, impaired auditory startle response and corresponding neuropathological findings) [The latter were already observed in an oral 90 -day study and might not represent a specific developmental effect, but rather systemic toxicity.]

Findings on fertility / developmental toxicity are assumed to be due to a non-human relevant mechanism. Further studies are currently running to evalute this endpoint.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

690 mg/kg bw/day

Species:

rat

Quality of whole database:

NOAEL converted based on DEA content of 5.5%.

Additional information

There are no data for the registered substance itself.

The source substance TEA was administered by gavage to 10 rats per sex and group at doses of 100, 300, and 1000mg/kg in a study according to OECD guideline 421 (6). Food consumption, body weight, clinical signs, mating and reproductive performance (including determinations of the number of implantations and the calculation of the post-implantation loss in females) were examined in parental animals. At necropsy, animals were assessed for gross pathology and selected organs were weighed and examined histopathologically. In pups, bodyweight, viability and macroscopic changes were recorded. At necropsy on PND 4, all pups were examined macroscopically for external and visceral findings. At the high dose of 1000 mg/kg bw/day, a decreased number of implantation sites, increased post-implantation loss and a lower average litter size were observed. No adverse effects were observed regarding reproductive performance, fertility or systemic toxicity at any dose level. Thus, the NOAEL for systemic toxicity as well as for reproductive performance and fertility in parental animals was established at 1000 mg/kg bw/day, and the NOAEL for prenatal developmental toxicity was determined to be 300 mg/kg bw/day.

With the source substance DEA, CAS 111-42-2, an extended One-Generation Reproductive Toxicity Study (EOGRTS) according to OECD TG 443 was performed, including cohorts 2A, 2B, and 3, but without the extension to an F2-generation. To assess the mode of action, additional parameters were investigated, e.g., the level of the essential nutrient choline in different tissues of the F0 and F1 pups (plasma and liver, respectively), as well as the platelet-activating factor (PAF). DEA was administered to groups of 30 male and 30 female Wistar rats (F0 parental generation) as a solution to the drinking water at concentrations of 100, 300 and 1000 ppm. This corresponds to app. 13, 38, and 128mg/kg bw. At least 16 days after the beginning of treatment, F0 animals were mated to produce a litter (F1 generation). Pups of the F1 litter were selected (F1 rearing animals) and assigned to the different cohorts. The study terminated with the terminal sacrifice of the male and female animals of cohort 1B.

Systemic Toxicity

In the F0 and F1 generation, DEA produced distinct effects on liver, kidney, and the hematopoietic system, comparable to those seen in repeated dose studies. The kidney and liver weights were dose-dependently increased in mid and high dose animals. The increase of platelet counts in mid and high dose males as well as the shortened prothrombin time (Hepatoquick’s test) in males and females of these groups, indicated a dysregulation of the coagulation homeostasis. In high dose animals, additionally an increase in ALP and AST was detected. Mid and high dose animals exhibited a higher urine volume and decreased water consumption. Toxic effects on liver and kidney were also confirmed by histopathological findings (centrilobular hypertrophy and lipid vacuoles in the liver, de- / regeneration and mineral depositions in the kidneys). Microcytic aneamie was detected in high dose animals. Additionally, the body weight was reduced in high dose animals and, less severely, in the mid dose. Consequently, the NOAEL for general toxicity was set to 100ppm (13mg/kg).

Fertility

Impaired fertility was observed in high dose animals as an increase in gestation time and a higher number of cannibalized pubs, as well as a prolonged estrous cycle in F1 females. The NOAEL for fertility was thus 300ppm (38mg/kg)

Developmental Toxicity

High dose F0 females had fewer implantation sites, which corresponded to a lower number of pubs in this group. Also, survival of pubs was decreased in the high dose. Histopathologically, fewer follicles were seen in high dose F1 females, while high dose F1 males displayed feminization of the mammary glands as well as vacuolization in the ductus deference (2 males) and tubular degeneration (1 male). Further changes in reproductive organs and developmental delays are considered secondary to the reduced body weight in the affected animals

High dose F1 animals displayed an impaired auditory startle response and corresponding neuropathological findings. In addition, increased T4 values were noted in adult and adolescent males and females at 1000 ppm as well as newborn and weanling females at 100 and 300 ppm (no high dose females available for measurement), although there was no associated change in TSH levels.

The NOAEL for developmental toxicity was 300ppm (38mg/kg).

Choline Measurements

Blood choline concentrations were measured in F1 animals at the age of 13 weeks. A dose dependent decrease was detected in all exposure groups, though the maximum of choline depletion seems to have been reached at the mid dose. No significant further reduction occurred in high dose animals.

Liver samples were analyzed in the F1 generation on PND4, 22, and 90. There was no clear difference between treated and control animals on PND 4. In 22-days old animals, choline content was dose-dependently reduced. Minimum concentration of app. 1/2 that of the control animals was already reached after treatment with 300ppm. In 90-days old animals, all doses led to a drastic reduction of choline content in the liver. The minimum was already reached in the 100ppm group. Higher doses did not result in a further reduction of choline levels.

As a summary, the following NOAELs were obtained:

General toxicity F0/F1: 100ppm / 13mg/kg (liver, kidney, hematopoietic system)

Fertility: F0/F1: 300ppm / 38mg/kg (increased gestation time, prolonged estrous cycle)

Developmental Toxicity: 300ppm / 38mg/kg (Lower number of implants, reduced pub survival, feminization of male mammary glands, effects on testes and ovaries, impaired auditory startle response and corresponding neuropathological findings) [The latter were already observed in an oral 90 -day study and might not represent a specific developmental effect, but rather systemic toxicity.]

Effects on developmental toxicity

Description of key information

OECD 414 (RA to DEA): No malformation after oral, dermal, or inhalation exposure. Increased post-implantation loss after oral exposure (no data in the remaining studies) at 200mg/kg

OECD 421 (RA to TEA): increased post-implantation loss at 1000mg/kg (BASF 2010)

OECD 443 (RA to DEA): The following NOAELs were obtained (BASF 2018):

- General toxicity F0/F1: 100ppm / 13mg/kg (liver, kidney, hematopoietic system)

- Fertility: F0/F1: 300ppm / 38mg/kg (increased gestation time, prolonged estrous cycle)

- Developmental Toxicity: 300ppm / 38mg/kg (Lower number of implants, reduced pub survival, feminization of male mammary glands, effects on testes and ovaries, impaired auditory startle response and corresponding neuropathological findings) [The latter were already observed in an oral 90 -day study and might not represent a specific developmental effect, but rather systemic toxicity.]

Findings on fertility / developmental toxicity are assumed to be due to a non-human relevant mechanism. Further studies are currently running to evalute this endpoint.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

360 mg/kg bw/day

Species:

rat

Quality of whole database:

NOAEL based on studies for TEA and DEA (see reproductive toxicity for details). Dose addition assumed. TEA (content of 39.2%) is app. 8 times less toxic than DEA (content of 5.5%). Conversion of the TEA NOAEL (300mg/kg) and the DEA NOAEL (38mg/kg) results in the combined NOAEL for the registered substance of 360mg/kg. (300 / 38 = 7.9 => 39.2% TEA is equal to a hypothetical DEA concentration of 5% (39.2% / 7.9). 38mg/kg / 10.5 (hypothetical DEA concentration) * 100 = 360mg/kg.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

200 mg/m³

Species:

rat

Quality of whole database:

No NOAEC conversion was performed. No malformations were seen in an oral OECD 414 study, but developmental toxicity occured in the corresponding OECD 443. These effects can also not be excluded after inhalation exposure.
Since all developmental effects occur only at doses with maternal / systemic toxicity, the risk assessment is based on these effects rather than fertility / dev. tox.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 500 mg/kg bw/day

Species:

rat

Quality of whole database:

No NOAEC conversion was performed. No malformations were seen in an oral OECD 414 study, but developmental toxicity occured in the corresponding OECD 443. These effects can also not be excluded after inhalation exposure.
Since all developmental effects occur only at doses with maternal / systemic toxicity, the risk assessment is based on these effects rather than fertility / dev. tox.

Additional information

There are no data for the registered substance itself.

DEA was administered dermally to pregnant CD rats from gestation days 6 to 15 at doses of 0, 150, 500 and 1500 mg/kg bw/day in a study performed similarly to OECD guideline 414 (7). At 500 and 1500 mg/kg bw/day moderate and severe skin irritation was caused, respectively. Maternal body weight gain was decreased in the 1500 mg/kg bw. Absolute and relative kidney weights were increased at 500 and 1500 mg/kg bw/day. Haematological effects including anaemia, abnormal red cell morphology (poikilocytosis, anisocytosis, polychromasia), and decreased platelet count were observed in all treatment groups. The 1500 mg/kg bw/day group also had increased lymphocytes and total leukocytes. In the fetuses, there were no effects of treatment on body weight or on incidence of external, visceral, or skeletal malformations/abnormalities. As a consequence of maternal toxicity, variations were increased in high dose pubs, e.g., poor ossification. Consequently, no NOAEL for maternal toxicity could be defined. The LOAEL was 150 mg/kg bw/day. Due to absence of visceral, skeletal, and gross malformations, the NOAEL for teratogenicity was >1500 mg/kg bw/day.

In an inhalation study according to OECD guideline 414, pregnant female Wistar rats were exposed to 0, 0.01, 0.05, and 0.2 mg/l of DEA aerosol in a head/nose exposure system for 6 h/day on days 6 through 15 post coitum (7). Eight of the 21 high dose animals had bloody vaginal discharge after exposure on day 14 p.c. At this dose level, a markedly increased number of fetuses with skeletal variations (mainly rudimentary cervical rib(s)) were recorded as a consequence of maternal toxicity. No substance-related teratogenic effects were detected in any group. The NOAEC for maternal toxicity was 0.05 mg/l, the NOAEC for teratogenicity was >0.2 mg/l.

In a non-guideline study, 12 time-mated pregnant rats per group were treated daily with 0, 50, 125, 200, 250, 300 mg/kg DEA by oral gavage from gestation day 6 through 19 (9). The females were allowed to litter, and development of the pubs was assessed until postnatal day 21. All females exposed to 300mg/kg were killed prematurely due to humane reasons. No further data on this group is therefore reported. One female receiving 250mg/kg was found dead on GD15. Another one of the same group was killed in extremis on GD21. In the 200mg/kg group, one female was euthanized while attempting to deliver 15 pubs, all of which were found dead in utero at necropsy. Reduced maternal body weight and weight change, as well as reduced feed intake, were noted at >= 200 mg/kg bw/day. At necropsy on PND 21, maternal absolute kidney weights were increased in rats treated with 125 mg/kg DEA and above, indicating persistence of DEA-induced toxicity for up to about 3 weeks after cessation of exposure. Exposure to 50 mg/kg bw/day was not associated with any significant maternal toxicity during or after the treatment period. Hematological parameters were not evaluated in this study. In rats treated with 200 and 250mg/kg, the post-implantation loss was increased to 17% and 51%, respectively, as compared to 2% in the control animals. Four of the remaining nine females treated with 250mg/kg had 100% post-implantation loss. Additionally, the number of live pubs per litter was dose dependently decreased on PND 0 to 55% in rats treated with 250mg/kg, but the difference did not reach statistical significance. From PND 0-4, postnatal mortality occurred in 0, 0.6, 1.8, 2.8, and 13.4% of pups /litter in the control - 250 mg/kg bw, respectively, being statistically significant at >= 125 mg/kg bw. For most pups that died postnatally, there was no milk found in the stomach, indicating unsuccessful nursing. After culling on PND 7, survival was identical in all groups. Pup body weight was reduced at >= 200 mg/kg bw/day, with females being more affected than males. When expressed as a percentage of control weight, pup body weight reductions were most pronounced during the early postnatal period. There were no substance-related morphological findings. Consequently, the NOAEL for maternal and developmental toxicity was 50 mg/kg bw/day.

DEA was administered dermally to pregnant New Zealand White rabbits from gestation days 6 through 18 at doses of 0, 35, 100 and 350 mg/kg bw/day in a study performed similarly to OECC 414 (7). High dose animals showed marked skin irritation, reduced food consumption, and color changes in the kidneys but no hematological changes. Body weight gain was reduced at and above 100 mg/kg bw/day. There was no impairment of gestational parameters. No evidence of developmental toxicity was observed at any dose level, especially, there were no apparent effects of treatment on the incidences of external, visceral, or skeletal abnormalities. Consequently, the NOAEL for maternal toxicity was 35 mg/kg bw/day, the NOAEL for prenatal developmental toxicity including teratogenicity was >350 mg/kg bw/day.

Further effects of DEA after oral administration observed in an OECD 443 study are described in the section on Reproductive toxicity / fertility.

Mode of Action Analysis / Human Relevance Framework

It has been discovered mainly in the context of carcinogenicity that ethanolamines adversely affect choline metabolism. It is possible, that the pre- and postimplantation losses are also mediated by effects on choline homeostasis rather than through a direct embryo toxicity. These effects may be inhibition of cholin-uptake in the liver, subsequent perturbation of choline-homeostasis, with subsequent impairment of C1-metabolism, DNA-methylation, lipid metabolism, and intercellular communication. Choline metabolism is connected to Phosphatidylcholine and Betaine. The latter is reported to be central for the synthesis of SAM (S-Adenosyl-Methionine), a principle methylating agent for biosynthetic pathways and maintenance of critical gene methylation patterns (17; 18).

DEA and TEA both inhibit the uptake of [3H]-choline in cultured CHO cells, with estimated EC50 values of 0.2 mM for DEA and 1.1 mM for TEA (17; 19).

DEA decreased gap junctional intracellular communication in primary cultured mouse and rat hepatocytes, induced DNA hypomethylation in mouse hepatocytes, decreased phosphatidylcholine synthesis, and increased S-phase DNA synthesis in mouse hepatocytes, but had no effect on apoptosis. All of these effects were mediated by the inhibition of choline sequestration, and were prevented with choline supplementation. No such effects were noted in human hepatocytes in vitro (20).

TEA decreases the hepatic levels of Phosphatidylcholine and Betaine, the primary oxidation product, by 35-42% and 26-29%, respectively, when given dermally for 3 weeks (5days per week) to female B6C3F1 mice at a dose of 1000mg/kg in two independent experiments (8). Choline concentration was not significantly decreased by 13-35%. At lower concentrations of 100 and 300mg/kg, there was a hint towards lower Phosphatidylcholine concentrations, which did not reach statistical significance. No changes of these parameters were observed under the same conditions at 250mg/kg in F344-derived rats. Since no higher doses, this does not infer a difference between rats and mice, nor does it contradict a relation between choline depletion and reproductive effects, since effects in either case were only observed at doses > 300mg/kg.

In the OECD 443 on DEA, there was a clear reduction in choline concentrations in the blood and liver samples, though the maximum effect was already reached at 300ppm. Additional mechanistic work will be conducted by the consortium members to further corroborate the link between reproductive effects observed for DEA as a secondary effect of an impairment choline homeostasis.

Justification for classification or non-classification

The registered substance contains > 3 % DEA (CAS # 111 -42 -2) which is classified as Repro Cat. 2, H361fd. Thus the registered substance also has to be classified with Repro Cat. 2, H361fd.