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Key value for chemical safety assessment

Effects on fertility

Additional information

For N-Methyldiethanolamin (mDEA) givendailyas an aqueous solution to groups of 10 male and 10 female Wistar rats (F0 animals) by stomach tube at doses of 0, 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) effects on implantation are reported. Clinical signs of toxicity were seen at the high dose level (1000 mg/kg bw/d) such as decreased food consumption in females during lactation and decreased body weight gain in males and females, resulting in reduced terminal body weights in both sexes. Reduced terminal body weights were also seen at the mid dose level (300 mg/kg bw/d). Total litter loss in 4 females, undelivered pups, insufficient lactation behavior, increased duration of gestation, decreased number of implantation sites, increased postimplantation loss and decreased number of delivered pups, reduced pup viability, decreased pup body weights and weight gains were seen exclusively at the high dose level (1000 mg/kg bw/d), i.e. only in the presence of parental toxicity.Pathology revealed increased liver weights at all dose levels, however without any morphological correlate. These findings were therefore assessed as being an adaptive phenomenon, but not an adverse effect. There were no further treatment-related lesions detected, especially, there were no weight or substance-related pathomorphological effects on testes, epididymides, and ovaries present. In conclusion, the administration of N-Methyldiethanolamin at dose levels of 1000 and 300 mg/kg bw/d caused toxic effects on body weight. The NOAEL (no observed adverse effect level) for general, systemic toxicity was therefore 100 mg/kg body weight/day for the F0 parental males and females. The NOAEL for reproductive performance and fertility was 300 mg/kg body weight/day for the F0 parental rats based upon finding such as litter loss, insufficient lactation behavior, and increased duration of gestation.The NOAEL for developmental toxicity was 300 mg/kg body weight/day, based on findings such as reducedviability index andreduced postnatal offspring weight gain.Thus, effects on reproductive parameters were affected only in the presence of parental toxicity.

 

No two-generation study is available with MDEA. For the structural analogue monoethanolamine (MEA) reproductive toxicity was investigated in a two-generation study with rats. For this structural analogue 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 . 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.

 

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.

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 intended to be performed 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 Methyldiethanolamine 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.

 

Smythet 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

 


Short description of key information:
A screening reproduction/developmental toxicity study with MDEA in the rat by oral gavage has been performed. The NOAEL for reproductive performance and fertility was 300 mg/kg body weight/day for the F0 parental rats based upon findings such as litter loss, insufficient lactation behavior, and increased duration of gestation. The NOAEL for parental toxicity was set at 100 mg/kg bw/day, based on body weight loss in both sexes. Thus, reproduction toxicity was only seen in the presence of parental toxicity.
Furthermore, for the structural analogue MEA, a two-generation reproduction toxicity study is available. Under the conditions of that study (performed with MEA HCl), the NOAEL for systemic toxicity and fertility / reproductive performance in parental F0 and F1 Wistar rats was 300 mg/kg bw/day. The NOAEL for pre-and postnatal developmental toxicity in their offspring was 1000 mg/kg bw/day. MEA does not need to be classified as a reproduction toxicant based on a weight of evidence approach using this two-generation study and the three available developmental toxicity studies with MEA. Based on the structural similarity, MDEA is also not considered a reproductive toxicant.

Effects on developmental toxicity

Description of key information
A screening reproduction/developmental toxicity study with MDEA in the rat by oral gavage has been performed. The NOAEL for developmental toxicity was 300 mg/kg bw/day based on findings such as reduced viability index and reduced postnatal offspring weight. The NOAEL for parental toxicity was set at 100 mg/kg bw/day, based on body weight loss in both sexes. Thus, developmental toxicity was only seen in the presence of parental toxicity.
In a prenatal developmental toxicity study, pregnant female rats were dermally exposed to MDEA on gestation days 6 to 15. The maternal and developmental NOAELs were established to be 250 and 1000 (the highest dose tested) mg/kg bw/day, respectively.
Additional information

In a reproduction/developmental screening study (performed according to OECD guideline 421), MDEA was given daily as an aqueous solution to female and male Wistar rats (F0 animals) by oral gavage at doses of 100, 300 and 1000 mg/kg bw/day (BASF, 2010). The duration of treatment covered pre-mating period of 2 weeks and a mating period (max. of 2 weeks) in both sexes and the entire gestation period as well as 4 days of lactation in females. Parental animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the post-implantation loss in all F0 females. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed one PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed and examined macroscopically for external and visceral findings at necropsy. All F0 parental animals were sacrificed and assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

Clinical signs of toxicity were seen at the highest dose level (1000 mg/kg bw/day) such as decreased food consumption in females during lactation and decreased body weight gain in males and females, resulting in reduced terminal body weights in both sexes. Reduced terminal body weights were also seen at the mid dose level (300 mg/kg bw/day).Pathologyrevealed increased liver weights at all dose levels, however without any morphological correlate. These findings were therefore assessed as being an adaptive phenomenon, but not an adverse effect. There wereno further treatment-related lesions detected, especially, there were no weight or substance-related pathomorphological effects on testes, epididymides, and ovaries present.

Concerning reproductive parameters, total litter loss in 4 females, undelivered pups, insufficient lactation behavior, increased duration of gestation, decreased number of implantation sites, increased post implantation loss and decreased number of delivered pups, reduced pup viability, decreased pup body weights and weight gains were seen exclusively at the high dose level (1000 mg/kg bw/day), i.e. only in the presence of parental toxicity.

In conclusion, the NOAEL for general, systemic toxicity was 100 mg/kgbw/day for the F0 parental males and females.The NOAEL for reproductive performance and fertility was 300 mg/kg bw/day for the F0 parental rats based upon litter loss, insufficientlactation behaviorand increased duration of gestation. The NOAEL for developmental toxicity was 300 mg/kg bw/day, based on reducedviability index andreduced postnatal offspring weight gain.

In a prenatal developmental toxicity study, pregnant CD rats (25/dose) were dermally exposed to 0, 250, 500 or 1000 mg/kg bw/day of MDEA on gestation day 6 -15 for 6 hours/day under occlusive conditions (Leung and Ballantyne, 1998). The NOAEL for maternal toxicity was determined to be 250 mg/kg bw/day, above which local irritant reactions at the application site were observed (including exfoliation, excoriation, crusting, ecchymoses, and necrosis). In addition, high dose dams had decreased erythrocyte counts, hemoglobin and hematocrit. There were no effects on any gestational parameters, and no increases in the total number of malformations or variations (external, visceral, or skeletal). Thus, the NOAEL for developmental toxicity was established at 1000 mg/kg bw/day, the highest dose tested.

In addition to the available dermal study with MDEA, read across with the structure analogue MEA is proposed to provide information on a second species. In the rabbit study with MEA exposure was via the dermal route to 0, 10, 25, and 75 mg/kg/day (protocol comparable to OECD 414). 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 (Liberacki et al, 1996).

Justification for classification or non-classification

Based on the available data, MDEA 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.