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Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Additional information

No substance-specific data on the reproductive toxicity of tris[(2 -hydroxyethyl)ammonium] citrate are available. However, according to Article 13 of the REACH legislation, in case no appropriate animal studies are available for assessment, information should be generated whenever possible by means other than vertebrate animal tests, i. e. applying alternative methods such as in vitro tests, QSARs, grouping and read-across.

Tris[(2 -hydroxyethyl)ammonium] citrate is a salt of monoethanolamine and citric acid and is expected to dissociated into the respective monoethanolammonium cation and dihydrogen citrate anion(which may subsequently undergo (partial) dissociation to monohydrogen citrate and citrate anions)upon uptake by the body.

Therefore it is considered to be acceptable to derive lacking information on toxicological properties of tris[(2 -hydroxyethyl)ammonium] citrate by read-across from its starting materials. Citric acid is an important intermediate of the Krebs cycle (also known as citric acid cycle), and therefore occurs naturally as a metabolite in virtually all living organisms. It is also used as a natural food preservative and a food additive. The average daily intake (ADI) is not limited according to the evaluation ofJoint FAO/WHO Expert Committee on Food Additives (1973), which concluded that the substance is non-hazardous to men. Therefore the toxicological behavior of tris[(2-hydroxyethyl)ammonium] citrate is expected to be governed primarily by the toxicity of monoethanolamine. Nevertheless available data on citric acid have also been taken into account.

Monoethanolamine hydrochloride was tested in an oral two-generation reproduction toxicity study with rats according to OECD Guideline 416 under GLP (BASF AG, 2009). The test substance was administered to groups of 25 male and 25 female rats (F0 parental generation) in diet at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. At least 75 days after the beginning of treatment, F0 animals were mated to produce a litter (F1 generation).After weaning of F1 pups the F0 generation parental animals were terminated. 25 male and 25 female F1 pups from each dose group were chosen to be F1 generation parental animals. They were treated with the test substance at the same dose levels up to about one day before they were terminated. At least 75 days after assignment of the F1 generation parental animals, the males and females were generally mated and the females were allowed to litter and rear their pups (F2 generation pups) until Day 4 (standardization) or 21 post-partum. Shortly after the F2 generation pups had been weaned, the F1 generation parental animals were terminated. All sacrificed F0 and F1 animals were necropsied and assessed by gross pathology and histopathology.

At 1000 mg/kg bw/day, signs of systemic toxicity were observed in parental females, manifested as reduced food consumption and/or body weight gain during gestation and lactation. In the mid and high dose F1 animals statistically significant increases of absolute and relative kidney weights were observed; however, these increases were not accompanied by histopathological changes

Decreased numbers of implants and increased resorption rates were observed in F0 and F1 females of 1000 mg/kg bw/day dose group, as well as significantly smaller litters. Absolute and relative weights of epididymides and cauda epididymidis were decreased in high-dose F0 and F1 males, and prostate weight and the number of homogenization resistant caudal epididymal sperm was slightly, but significantly reduced in high-dose F0 males. These findings occurred in the absence of histopathological changes. There were no changes in estrous cycle, mating behavior, conception, gestation, parturition, lactation and weaning. No adverse developmental changes occurred at any dose level. The test substance did not adversely influence pup viability, body weight, sex ratio and sexual maturation.

Based on the results of the study, the NOAEL (no observed adverse effect level) for systemic toxicity and reproductive toxicity 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. As adverse effects on fertility occurred only at dose levels causing parental toxicity, classification of monoethanolamine for reproductive effects is not warranted.

No conventional studies on reproductive toxicity are available for citric acid; however, a widespread use of the substance and the lack of reports on possible reproductive effects allow to conclude with a high degree of certainty that the substance is not a reproductive toxicant.

Respectively, tris[(2 -hydroxyethyl)ammmonium] citrate is also considered not a reproductive toxicant. Choosing the NOAEL of 300 mg/kg bw/day as a point of departure, and applying a correction for molecular weight, a NOAEL of 300×(375.37/3)/61.08 = 614.6 mg/kg bw/day can be calculated for tris[(2-hydroxyethyl)ammonium] citrate. No DNEL for reproductive toxicity needs to be derived.

Short description of key information:
Based on the results of the two-generation reproductive toxicity study with rats with a read-across candidate monoethanolamine and the lack of reprotoxic properties of citric acid, tris[(2-hydroxyethyl)ammonium] citrate is not considered a reproductive toxicant.

Effects on developmental toxicity

Description of key information
Based on the results of the three developmental toxicity study with rats with a read-across candidate monoethanolamine and the lack of developmental toxicity for citric acid, tris[(2-hydroxyethyl)ammonium] citrate is not considered a developmental toxicant. 
Effect on developmental toxicity: via oral route
Dose descriptor:
NOAEL
921.8 mg/kg bw/day
Effect on developmental toxicity: via dermal route
Dose descriptor:
NOAEL
460.9 mg/kg bw/day
Additional information

No substance-specific data on the developmental toxicity of tris[(2 -hydroxyethyl)ammonium] citrate are available. However, according to Article 13 of the REACH legislation, in case no appropriate animal studies are available for assessment, information should be generated whenever possible by means other than vertebrate animal tests, i. e. applying alternative methods such as in vitro tests, QSARs, grouping and read-across.

Tris[(2 -hydroxyethyl)ammonium] citrate is a salt of monoethanolamine and citric acid and is expected to dissociated into the respective monoethanolammonium cation and dihydrogen citrate anion (which may subsequently undergo (partial) dissociation to monohydrogen citrate and citrate anions) upon uptake by the body.

Therefore it is considered to be acceptable to derive lacking information on toxicological properties of tris[(2 -hydroxyethyl)ammonium] citrate by read-across from its starting materials. Citric acid is an important intermediate of the Krebs cycle (also known as citric acid cycle), and therefore occurs naturally as a metabolite in virtually all living organisms. It is also used as a natural food preservative and a food additive. The average daily intake (ADI) is not limited according to the evaluation ofJoint FAO/WHO Expert Committee on Food Additives (1973), which concluded that the substance is non-hazardous to men. Therefore the toxicological behavior of tris[(2-hydroxyethyl)ammonium] citrate is expected to be governed primarily by the toxicity of monoethanolamine. Nevertheless available data on citric acid have also been taken into account.

Three reliable developmental toxicity studies, two with rats and one with rabbits, are available for monoethanolamine. In the first study, performed according to OECD Guideline 414 and under GLP (BASF AG, 1994; Hellwig and Liberacki, 1997) monoethanolamine was administered by gavage at dose levels 0, 40, 120, 450 mg/kg bw/day to 40 pregnant rats on days 6 - 15 of gestation. Statistically significant reduction in food consumption, mean body weight and body weight gain were observed at the highest dose level. No signs of developmental toxicity occurred up to and including the highest dose level, especially no substance-induced teratogenic effects were observed neither in the fetuses nor in the pups. Furthermore, there were no indications for any substance-related growth retardations. All skeletal malformations, variations, or retardations which occurred did not show a clear dose-response relationship, can be found at comparable or even higher rates within the historical control and were considered not to be related to the test substance. Based on the results of the study, the NOAEL for developmental effects was set at 450 mg/kg bw/day, while the NOAEL for maternal toxicity was set at 120 mg/kg bw/day.

Another study with rats and the study with rabbits were performed using dermal route of administration. In the study with rats (Liberacki, 1996), performed according to a protocol comparable to OECD guideline 414, groups of 30 -45 pregnant Sprague-Dawley rats were exposed dermally to 0, 10, 25, 75 and 225 mg/kg/day of monoethanolamine for approximately 6 hr/day on Days 6 through 15 of gestation. Animals were sacrified at day 21 of gestation. At 225 mg/kg bw/day increased incidence of skin irritation and a statistically significant decrease of the body weight gain was observed. No significant dermal irritation or lesions were observed among rats administered lower doses of monoethanolamine. There were no differences in pregnancy rate, number of corpora lutea, number of implantations, resorptions, litter size, number of dead fetuses, fetal sex ratio, fetal body weight, or gravid uterine weight among any of the dose groups when compared to controls. There were no treatment-related increases in the incidence of variations or malformations observed externally, viscerally or at skeletal examination by individual category, or in total variations or malformations when compared to controls. 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 study with rabbits (Liberacki et al, 1996; Neeper-Bradley and Kubena, 1993), pregnant New Zealand White rabbits (15/group) were exposed dermally to 0, 10, 25, and 75 mg/kg/day of monoethanolamine for approximately 6 hr/day on Days 6 through 18 of gestation. Animals were sacrificed on day 29 of gestation. Signs of skin irritation were observed in mid and high dose group, severe at the highest dose level. No treatment-related effects were observed on reproductive parameters including pregnancy rate, number of corpora lutea, number of implantations, resorptions, litter size, number of dead fetuses, fetal sex ratio, fetal body weight, or gravid uterine weight at any dose level when compared to controls. There were no statistically or biologically significant treatment-related differences in the incidence of any fetal variation or malformation, or in the number of malformed fetuses in any dose group. Based on the results of the study, 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.

Based on the results of all three studies, monoethanolamine is not a developmental toxicant. No conventional studies on developmental toxicity are available for citric acid; however, a widespread use of the substance and the lack of reports on possible developmental effects allow to conclude with a high degree of certainty that the substance is not a developmental toxicant.

Respectively, tris[(2 -hydroxyethyl)ammonium] citrate is also considered to be not toxic to development. Using the NOAEL of 450 mg/kg bw/day established in the oral developmental toxicity study and 225 mg/kg bw/day in the dermal developmental toxicity study with monoethanolamine as a starting point and applying a correction for molecular weight, a NOAEL for developmental toxicity of tris[(2 -hydroxyethyl)ammonium] citrate is calculated to correspond to 921.8 mg/kg bw/day for oral and to 460.9 mg/kg bw/day for dermal routes of exposure. As no adverse effects on development occurred even at dose levels far above doses causing maternal toxicity, no DNEL for develomental toxicity will be derived.

Justification for classification or non-classification

Based on the read-across with the starting materials monoethanolamine and citric acid, classification of (2 -hydroxyethyl)ammonium dihydrogen citrate for reproductive and developmental toxicity is not warranted according to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Additional information