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Key value for chemical safety assessment

Effects on fertility

Additional information

Please see justification for classification and non-classification for details.


Short description of key information:
No two-generation reproduction toxicity study is available for TEA. MEA is a structural analogue of TEA. Under the conditions of a two-generation reproduction toxicity study with MEA HCl, the NOAEL for systemic toxicity and fertility, reproductive performance in parental F0 and F1 Wistar rats is 300 mg/kg bw/day. The NOAEL for pre-and postnatal developmental toxicity in their offspring is 1000 mg/kg bw/day.
Furthermore, in a screening reproduction/developmental toxicity study (OECD 421) with TEA in rats, the NOAEL for systemic toxicity as well as for reproductive performance and fertility in parental animals was established at 1000 mg/kg bw/day, the highest dose tested. The NOAEL for postnatal toxicity in the offspring was 1000 mg/kg bw/day, whereas the NOAEL for prenatal developmental toxicity was determined to be 300 mg/kg bw/day based on decreased numbers of implants and delivered pups, and an increased postimplantation loss.

Effects on developmental toxicity

Description of key information
In an oral screening reproduction/developmental toxicity study (OECD 421) with TEA in rats, the NOAEL for systemic toxicity and postnatal toxicity in the offspring was 1000 mg/kg bw/day, whereas the NOAEL for prenatal developmental toxicity was determined to be 300 mg/kg bw/day based on decreased numbers of implants and delivered pups, and an increased postimplantation loss.
In an oral Chernoff-Kavlok teratogenicity screening test, TEA did not produce any evidence of developmental or maternal toxicity in CD-1 mice. Therefore, the NOAEL for maternal toxicity and developmental toxicity was established at 1125 mg/kg bw/day.
As no complete developmental toxicity study (OECD guideline 414) is available for TEA, read across with the structural analogue MEA, for which developmental toxicity studies are available, is applied. Based on the results of the screening studies with TEA (oral route, rats and mice) and the available developmental toxicity studies with rats and rabbits (oral and dermal route of exposure) with MEA, TEA is not considered to be a developmental toxicant.
Additional information

In a reproduction/developmental toxicity screening study with TEA, performed according to OECD guideline 421, Wistar rats (10/sex/dose) were exposed by gavage to 0, 100, 300 or 1000 mg/kg bw/day during a premating period of 2 weeks and a mating period (max. 2 weeks) for both sexes, during approximately 1 week post-mating for males, and during the entire gestation period as well as 4 days of lactation for females. Food consumption, body weight, clinical signs, mating and reproductive performance (including determinations of the number of implantations and the calculation of the postimplantation 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 postimplantation 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, the NOAEL for postnatal toxicity in the offspring was 1000 mg/kg bw/day, and the NOAEL for prenatal developmental toxicity was determined to be 300 mg/kg bw/day (BASF AG, 2010).

In a Chernoff-Kavlok teratogenicity screening test, CD-1 mice were exposed to by gavage in 3 phases: 1) 3 virgin females were exposed to 10, 100 of 1000 mg TEA/kg bw/day during 5 consecutive days; 2) 2 -4 mated females were exposed to 600, 1200, 2400, 4800 or 9600 mg TEA/kg bw/day on gestation days (GD) 6 -15; 3) 50 mated females were exposed to 1125 mg TEA/kg bw/day on GD 6 -15. In the main study (phase 3), exposure to TEA did not produce any evidence of developmental or maternal toxicity. Therefore, the NOAEL for maternal toxicity and developmental toxicity was established at 1125 mg/kg bw/day (Environmental Health Research & Testing Inc., 1987).

As no complete developmental toxicity study (OECD guideline 414) is available for TEA, read across with the structural analogue MEA, for which developmental toxicity studies are available, is applied.

In a GLP-compliant prenatal developmental toxicity study with rats, performed according to OECD guideline 414 (BASF AG, 1994) pregnant Wistar rats were exposed to the structure analogue MEA by gavage at dose levels 0, 40, 120, 450 mg/kg bw/day on days 6 - 15 of gestation. Signs of maternal toxicity were observed at the highest dose, manifested as reduced food consumption, lower mean body weights and impaired body weight gain. No reproductive and developmental toxicity parameters were affected. The NOAEL for developmental effects was thus established to correspond to 450 mg/kg bw/day; the NOAEL for maternal toxicity was 120 mg/kg bw/day.

 

In another comparable to guideline prenatal developmental toxicity study (Liberacki, 1996) rats and rabbits were exposed.

Pregnant Sprague-Dawley rats were exposed dermally to 0, 10, 25, 75 and 225 mg/kg bw/day of MEA. Rats administered 225 mg MEA/kg bw/day exhibited a treatment-related increased incidence of skin irritation and the body weight gain was significantly decreased during the exposure period.Despite maternal effects observed among dams in the high dose group, reproductive and developmental toxicity parameters among exposed rats were unaffected at all dose levels. The NOAEL for maternal toxicity was set at 75 mg/kg bw/day and the NOAEL for developmental toxicity was set at the highest dose level of 225 mg/kg bw/day.

In the rabbit study exposure was via the dermal route to 0, 10, 25, and 75 mg/kg/day of MEA. The rabbits in the mid and high dose group exhibited signs of skin irritation, severe at the highest dose level. No treatment-related effects were observed on reproductive and developmental toxicity parameters. The NOAEL for maternal toxicity was set at 10 mg/kg bw/day and the NOAEL for developmental toxicity was set at the highest dose level of 75 mg/kg bw/day.

In a preliminary study on the prenatal toxicity of MEA, female rats (10/dose) were exposed to 0, 50, 150, 300 or 500 mg/kg bw/day by gavage on gestation days 6 -15. Maternal toxicity was observed at the high dose only, and included reduced food consumption, impaired body weight gain, decreased total protein and albumin levels, and a thickened wall of the forestomach in 3 dams. No adverse effects on the fetuses occurred. Therefore, NOAELs for maternal toxicity and teratogenicity were established at 300 and 500 mg/kg bw/day (the highest dose tested), respectively (BASF AG, 1992).

Based on the results of the screening studies with TEA (oral route, rats and mice) and the available developmental toxicity studies with rats and rabbits (oral and dermal route of exposure) with MEA, TEA is not considered to be a developmental toxicant.

Justification for classification or non-classification

Based on the available data, TEA does not need to be classified for effects on fertility and developmental toxicity according to Annex I of Directive 67/548/EEC and according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

In a standard screening study to OECD TG 421 (BASF, 2010), Triethanolamine (TEA) was administered by gavage (vehicle water) to groups of 10 male and 10 female Wistar rats at dose levels of 0, 100, 300, or 1000 mg TEA/kg bw/day. At the highest dose level there was a statistically significant decrease in litter size and increase in post-implantation loss. The number of implantation sites was decreased by 20%, but this was not statistically significant. A reduction in maternal bodyweight gain during gestation is attributed to the smaller litter sizes in the high dose group. There were no treatment-related effects on postnatal survival or pup bodyweights. Although bodyweights in the high dose group were ca. 8% higher than control, this was not statistically significant and probably reflects, if anything, the smaller litter sizes.

 

For Monoethanolamine (MEA) a recent two generation reproduction toxicity study in Wistar rats with dietary MEA administration demonstrated clear NOAELs for systemic and reproductive toxicity including fertility at 300 mgMEA-HCl/kg bw/day. Only at the highest dose, 1000 mg/kg bw/day, were minor effects noted. Males at this high dose levels showed minor effects on fertility in the form of decreased absolute and relative weights of epididymides and cauda epididymidis and, in the F0 generation only, a significantly lower number of homogenization resistant caudal epididymal sperm compared to control. However, there was no histomorphological correlate of these findings in the organs, no effect upon testes or testicular sperm count, and no effect upon mating peformance. Females at this dose level revealed decreased numbers of implants and increased resorption rates resulting in smaller litters associated with indications of systemic toxicity. There was virtually no effect on the pre- and postnatal development of the progeny in both generations up to the limit dose level of 1000 mg/kg bw/day representing a clear NOAEL for developmental toxicity. In a previous repeated dose toxicity study, rats were administered 160 to 2,670 mg/kg bw/day MEA in the diet for 90 days. Deaths occurred at 1280 mg/kg bw/day and the liver and kidney weights were increased at 640 mg/kg bw/day. The NOAEL was 320 mg/kg bw/day (Smyth, 1951).

There are no data available to assess the potential effects of Diethanolamine (DEA) upon implantation.

The US National Toxicology Program conducted a prenatal developmental toxicity study by gavage administration of DEA to groups of 12 pregnant CD rats at doses of 0, 50, 125, 200, 250, or 300 mg/kg bw/d during GD 6–19 (Priceet al., 2005). The test substance was administered in water, and the pH of the dosing solutions was adjusted to 7.4±0.2 with hydrochloric acid. Dams were allowed to litter and raise their offspring to PND 21.

Dams in the high dose group showed signs of excessive toxicity and were euthanised by GD 15; the following discussion omits further mention of this group. The principal toxicity noted in the remaining dams was of a dose-related reduction in bodyweight (gain) during gestation compared to the control group. This was most noticeable at 250 mg/kg BW/d, in which animals had lost weight by GD 12 and didn’t start gaining weight until GD 15, after which weight gain maintained parity with the control. Animals in the 200 mg/kg bw/d group lost weight by GD 12, but weight gain maintained parity with the control thereafter. There were no differences in bodyweight (gain) between control and the other treatment groups.

There was a statistically significant increase in post-implantation loss in the groups that were administered 200 or 250 mg/kg bw/d, which in the higher dose group was also manifested as total loss of four litters in dams that survived until term, one of which consisted of all dead pups at PND 0 and the other three which consisted only of implantation sites. It is not clear from the publication as to whether the implantations were lost at an early or later stage. Two other dams in this group were either found dead on GD 15 or euthanised moribund on GD 21. Both had litters. One dam in the 200 mg/kg bw/d dose group was euthanised on GD 22 and had a litter of dead foetuses. There was a statistically significant increase in postnatal mortality during PND 0–4 in groups administered ≥125 mg DEA/kg bw/d.

Taken together, similar effects on pre- and/or post-implantation losses were observed for Mono-, Di- and Triethanolamine. Additionally, all three Ethanolamines show similar effects on choline-metabolism.

Ethanolamines inhibit the uptake of [3H]-choline in cultured CHO cells, with estimated EC50values of ~0.2 mM for DEA and ~1.1 mM for TEA (Lehmann-McKeeman and Gamsky, 1999; Stottet al., 2004). In comparison, the EC50for diisopropanolamine is ~0.5 mM (Stott and Kleinert, 2008).

 

For DEA various mechanisticin vitroandin vivostudies identified that choline depletion is the key event in hepatic carcinogenicity. 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 hepatocytesin vitro. Apparent differences in the susceptibility of two different mice strains (B6C3F1 > C57BL) were noted. B6C3F1 mice are extremely sensitive to non-genotoxic effects and are susceptible to spontaneous liver tumors. Moreover, chronic stimulation and compensatory adaptive changes of hepatocyte hypertrophy and proliferation are able to enhance the incidence of common spontaneous liver tumors in the mouse by mechanisms not relevant to humans (adapted from the DEA OECD SIAR, 2009).

For TEA it is reported that it decreases the hepatic levels of Phosphatidylcholine and Betaine, the primary oxidation product, when TEA is given dermally to female B6C3F1mice (Stott, 2004) at the high dose of 1000 mg/kg bw up to 26-42% indicating a disturbance. In this study by Stott et al. (2004) no changes on hepatic Phosphatidylcholine and Betaine were reported in F344-derived rats. However, only a single dose of 250 mg TEA/kg bw/day was tested in female rats for 3 weeks (5days/week). Higher doses of TEA applied orally as it has been done in the available OECD 421 might cause the same effects as observed in mice. Furthermore, a strain difference in rats’ sensitivity to choline depletion cannot be excluded. TEA also inhibited the ³H-choline uptake in vitro in Chinese hamster ovary cells. DEA is able to increase cell proliferation (probably via the same mechanism) in mice and rat hepatocytes whereas this effect is not observed in human hepatocytes.

It is possible that the effects of MEA, DEA and TEA on pre- and post-implantation may be mediated by effects on choline homeostasis (as described above) 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 (Stott et al. 2004; Zeisel and Blusztajn, 1994). 

Demonstration of a choline-dependency of the critical window for the observed effect on pre- and postimplantation would provide a basis for evaluating the Human relevance or non-relevance of these findings. Research is planned to clarify this and to allow a final evaluation and hazard assessment of the observed findings. Investigations are proposed to determine whether or not choline-supplementation rescues protects against pre- and postimplantation losses. A second step will be the elucidation of the mode-of-action. It has been accepted for DEA tumorigenicity, that the effects observed are caused by a non-genotoxic modulation of DNA-methylation. Such effects may also explain the observed effects on implantation. Significantly, this is important for the final evaluation of the Ethanolamines as this potential mode-of-action may display a species-specific effect with humans being resistant towards choline-deficiency and its consequences in rodents.

Further research has been proposed that may render additional information on the relevance to human. Therefore, it is proposed not to classify Triethanolamine at this time so that the additional research can be considered.

 

 

References

Lehman-McKeeman LD, Gamsky EA (1999). Diethanolamine inhibits choline uptake and phosphatidylcholine synthesis in Chinese hamster ovary cells.Biochem Biophys Res Commun262:600–604.

 

OECD SIDS (2009). Diethanolamine.

 

Smyth et al, (1951). Range-finding Toxicity Data: List IV.Arch.Hyg. Occup. Med.4: 119 - 122

 

Stott WT, Kleinert KM (2008). Effect of diisopropanolamine upon choline uptake and phospholipid synthesis in Chinese hamster ovary cells.Fd Chem Toxicol46:761-766.

 

Stott WT, Radtke BJ, Linscombe VA, Mar M-H, Zeisel SH (2004).Evaluation of the potential of triethanolamine to alter hepatic choline levels in female B6C3F1 mice.Toxicol Sci79:242-247.

 

Zeisel SH and Blasztajn JK (1994). Cholin and human nutrition.Ann. Rev. Nutr.14: 269-296