Registration Dossier
Registration Dossier
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EC number: 939-464-2 | CAS number: 121617-08-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
- Toxic effect type:
- dose-dependent
Effects on fertility
Description of key information
There are no studies on toxicity to reproduction performed on LAS TEA. The endpoint is addressed using read across from three analogues.
LAS Na (C10 -14):
LAS Na (C10 -14; CAS 69669-44-9) was fed for 84 days to 4 groups of weanling rats for two years (three generations) at doses of 14, 70, 350 mg/kg bw/day. No significant effects were observed even at the highest dose tested and the resulting NOAEL for the parental and both offspring generations was determined to be 350 mg/kg bw/day (0.5%) (Buehler et al., 1971).
TEA:
In a screening reproduction/developmental toxicity study (OECD 421) in rats, TEA was administered to male and female Wistar rats, orally by gavage, at dose levels of 0, 100, 300, 1000 mg/kg bw/day. 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 post-natal toxicity in the offspring was 1000 mg/kg bw/day, whereas the NOAEL for pre-natal developmental toxicity was determined to be 300 mg/kg bw/day based on decreased numbers of implants and delivered pups, and an increased post-implantation loss (BASF, 2010).
MEA:
MEA is a structural analogue of TEA. MEA HCl was given to rats in a diet at dose levels of 0, 300, 500, and 1000 mg/kg bw/day. Under the conditions of this study, 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 post-natal developmental toxicity in their offspring is 1000 mg/kg bw/day (BASF, 2009).
Link to relevant study records
- Endpoint:
- two-generation reproductive toxicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: read-across to Klimisch 1 GLP Guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
- Deviations:
- yes
- Remarks:
- Food consumption was not determined between days 14 and 21 after parturition
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- other: Crl:WI (Han)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Germany
- Age at study initiation: (P) 16 days
- Weight at study initiation: (P) Males: 162.1 (142.5 ¿ 186.5) g ; Females: 126.2 (110.6 ¿ 145.1) g;
- Fasting period before study: none
- Housing: housed individually in type DK III stainless steel wire mesh cages
- Diet: ground Kliba maintenance diet mouse/rat ¿GLP¿ meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland ad libitum
- Water: ad libitum
- Acclimation period: 16 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
- Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- DIET PREPARATION
The test substance (ethanolamine hydrochloride, EAH) was weighed and thoroughly mixed with a small amount of food. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer. Test diets were prepared at intervals, which guaranteed that the test substance in the diet remained stable throughout the feeding period.
- Details on mating procedure:
- - M/F ratio per cage: 1/1
- Length of cohabitation: over night
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged (how): individual - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The stability of EAH in the diet over 32 days at room temperature was investigated analytically before the beginning of the study. Homogeneity and concentration control analyses were carried out at the beginning and toward the end of the premating periods. At least one analysis of test substance preparations for female animals was carried out during the gestation and lactation periods.
The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. - Frequency of treatment:
- daily
- Remarks:
- Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
nominal in diet - No. of animals per sex per dose:
- 25
- Control animals:
- yes, plain diet
- Positive control:
- not done
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days and once daily on weekends
DETAILED CLINICAL OBSERVATIONS: Yes
BODY WEIGHT: Yes
- Time schedule for examinations: body weights of F0 and F1 parents were determined once weekly; during gestation and lactation F0 and F1 females were weighed on days 0, 7, 14 and 20 of gestation, and on days 1, 4, 7, 14 and 21 after birth.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule: once weekly (over a period of at least 6 days each) and weekly during gestation (days 0-7, 7-14, 14-20 post coitum; p.c.) and lactation periods (days 1-4, 4-7, 7-14 post partum; p.p.).
OTHER:
The F1 and F2 pups were sexed on the day of birth (day 0 p.p.) and weighed on days 1, 4, 7, 14, and 21 p.p. Their viability was recorded. At necropsy, all pups were examined macroscopically (including weight determinations of brain, spleen and thymus in one pup/sex/litter).
Serum concentrations of the test substance:
Blood samples were taken from all F0 and F1 parental animals of each sex and test group during week 10 of premating treatment and the plasma was analyzed for the concentration of Ethanolamine hydrochloride - Oestrous cyclicity (parental animals):
- Estrous cycle data were evaluated for F0 and F1 generation females over a three week period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.
- Sperm parameters (parental animals):
- Parameters examined in [all/P/F1] male parental generations:
motility, sperm head count, morphology - Postmortem examinations (parental animals):
- All F0 and F1 parental animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention was given to the reproductive organs. The liver, kidneys, adrenal glands, testes, epididmides. Cauda epididymis, prostate, seminal vesicles, ovaries, uterus, spleen, brain, pituitary gland and thyroid glands (with parathyroids) were weighed and the vagina, cervix uterie, uterus, ovaries, oviducts, left testis, left epididymis, seminal vesicles, coagulation glands, prostate, pituitary gland, adrenal glands, liver, kidneys, spleen, brain, thyroids (with parathyroids)and all gross lesions were fixed in an appropriate fixative, histologically processed and examined by light microscopy. From both ovaries (¿ovary 1¿ and ¿ovary 2¿) of F1 female animals (control and top dose), five sections were taken from the proximal and the distal part of the ovaries, at least 100 µm apart from the inner third of the ovary. All ovarian sections were prepared and evaluated for numbers of primordial and growing follicles.
As soon as possible after termination, one portion of the liver (lobus medialis) of each 10 dams per group was sampled to be analyzed for choline concentration. - Postmortem examinations (offspring):
- All pups with scheduled sacrifice (i.e. pups, which were culled on day 4 p.p., and pups, which were sacrificed on day 21 p.p. or subsequent days) were killed by means of CO2. The spleen and thymus of 1 pup/sex and litter from the F1 and F2 pups were weighed. All stillborn pups and all pups that died up to weaning were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.
- Statistics:
- see below
- Clinical signs:
- no effects observed
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- effects observed, treatment-related
- Reproductive performance:
- effects observed, treatment-related
- Dose descriptor:
- NOAEL
- Effect level:
- 300 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- other: fertility, reproductive performance and systemic toxicity; absolute and relative weights of epididymides and cauda epididymidis
- Clinical signs:
- no effects observed
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Sexual maturation:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- no effects observed
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- other: pre-and postnatal developmental toxicity; pup viability, body weight, sex ratio and sexual maturation.
- Dose descriptor:
- NOAEL
- Generation:
- F2
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- other: pre-and postnatal developmental toxicity; pup viability, body weight, sex ratio and sexual maturation.
- Reproductive effects observed:
- not specified
- Conclusions:
- The substance had an effect on the reproductive performance of the F0 and F1 animals when tested at the dose level of 1000 mg/kg bw.
- Executive summary:
In a two-generation reproduction study 2-aminoethanol (CAS 141-43-5) was administered to 25 Crl:WI (Han) rats/sex/dose in diet at dose levels of 0, 300, 500, and 1000 mg/kg bw/day. No test substance-related findings from clinical examinations or gross and histopathology were observed. At the high-dose level, absolute and relative weights of epididymides and cauda epididymidis were decreased and, in the F0 generation only, the number of homogenization resistant caudal epididymal sperm was slightly, but significantly reduced. However, histomorphological correlates for these findings were missing. At the same dose level food consumption and body weight gain of the dams (F0 and F1) was significantly decreased during gestation/lactation.
In the high-dose F0 and F1 generation, decreased numbers of implants and increased resorption rates resulted in significantly smaller litters, giving evidence for an adverse effect of the test compound on fertility and/or reproductive performance at high doses. All data recorded during gestation and lactation in terms of embryo-/fetal and pup development gave no indications for any developmental toxicity in the F1 and F2 offspring up to a dose level of 1000 mg/kg bw/d. The test substance did not adversely influence pup viability, body weight, sex ratio and sexual maturation. The NOAEL(no observed adverse effect level) for fertility, reproductive performance and systemic toxicity in parental F0 and F1 Wistar rats is 300 mg/kg bw/d.The NOAEL for pre-and postnatal developmental toxicityin their offspring is 1000 mg/kg bw/d.
- Endpoint:
- screening for reproductive / developmental toxicity
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: read-across to Klimisch 1 GLP guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
- Qualifier:
- according to guideline
- Guideline:
- other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Male and female Wistar rats, strain Crl:WI(Han), supplied by Charles River Laboratories, Research Models and Services, Germany GmbH, which were free from any clinical signs of disease, were used for the investigations. The females were nulliparous and non-pregnant at the beginning of the study. According to a written statement from the breeder, male and female animals were derived from different litters. This was necessary to rule out the possibility of sibling mating. These animals were used as F0 generation parental animals. All other animals used in this study (F1 generation pups) were derived from the supplier-provided animals.
During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
- During overnight matings, male and female mating partners were housed together in Makrolon type M III cages.
- Pregnant animals and their litters were housed together until PND 4.
Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment wooden gnawing blocks (Typ NGM E-022; supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria) were added. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured. The animals were housed in fully air-conditioned rooms in which central air conditioning guaranteed a range of temperature of 20-24 °C and a range of relative humidity of 30-70%. The air change rate was 15 times per hour. There were no or only minimal deviations from these limits. The day/night cycle was 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h.
The animal room was completely disinfected using a disinfector ("AUTEX" fully automatic, formalin-ammonia-based terminal disinfection) before use. Walls and floor were cleaned each week with water containing about 0.5% Incidin Extra N (supplied by Ecolab Deutschland GmbH, Hanau, Germany) and 0.5% Mikro-Quat (supplied by Ecolab GmbH & Co. OHG, Düsseldorf, Germany). The food used was ground Kliba maintenance diet mouse/rat ¿GLP¿ meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, which was available to the animals ad libitum throughout the study (from the day of supply to the day of or the day before necropsy). Drinking water was supplied from water bottles (ad libitum). The bedding used was Lignocel FS 14 fibres, dustfree bedding, supplied by SSNIFF, Soest, Germany.
The 45 male and 45 female rats were 9 weeks old when they arrived from the breeder. During an acclimatization period of about 6 days, animals with lowest and highest body weights were eliminated from the study and used for other purposes. The 40 male and 40 female animals included in the study were 10 weeks old at the beginning of treatment, and their body weights varied between:
- male animals: 357.7 g - 301.4 g
- female animals: 173.2 g - 205.8 g
The assignment of the animals to the different test groups was carried out using a randomization program, according to their weight four days before the beginning of the administration period (day -4). - Route of administration:
- oral: gavage
- Type of inhalation exposure (if applicable):
- other: not applicable
- Vehicle:
- water
- Details on exposure:
- The test substance solutions in drinking water were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification. The maximum period for which each preparation was used was 7 days. For the preparation of the administration solutions the test item was weighed in a graduated measuring flask depending on the dose group, topped up with drinking water and subsequently thoroughly shaken until completely dissolved.
The volume administered each day was 10 ml/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.
Analyses of the test substance preparations
The analyses were carried out at at Competence Center Analytics, BASF SE, Ludwigshafen, Germany. Analytical verifications of the stability of the test substance in drinking water for a period of 7 days at room temperature were carried out prior to the start of the study. Given that Triethanolamin rein is completely miscible with drinking water, solutions were considered to be homogenous without further analysis. Samples of the test substance solutions were sent to the analytical laboratory twice during the study period for verification of the concentrations. Of each sample, one additional reserve sample was retained. Details of the sampling schedule were recorded with the raw data. - Details on mating procedure:
- In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group.
The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "GD 0" and the following day "GD 1". - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The concentration of the test substance in the vehicle (drinking water) was checked by capillary electrophoresis (CE) with internal standard quantification, using a Beckman P/ACE MDQ automated capillary electrophoresis system including capillary oven and UV-detector.
- Duration of treatment / exposure:
- Premating period of 2 weeks and a mating period (max. 2 weeks) in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females.
- Frequency of treatment:
- Daily
- Details on study schedule:
- After the acclimatization period, the test substance was administered to the parental animals orally by gavage, once daily at approximately the same time in the mornings. Females in labor were not treated. The treatment lasted up to one day prior to sacrifice. The animals of the control group were treated with the vehicle (drinking water), in the same way. Males and females from the same dose group were mated after a 14 days premating period, overnight in a ratio of 1:1. The females were allowed to deliver and rear their pups until day 4 after parturition. Shortly after PND 4 the parental females were sacrificed and examined. Pups were sacrificed on PND 4 and gross necropsied. The male animals were sacrificed 36 days after the beginning of the administration, and examined.
- Remarks:
- Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
actual ingested - No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Positive control:
- None
- Parental animals: Observations and examinations:
- Mortality
A check for moribund or dead animals was made twice daily on working days or once daily on Saturdays, Sundays or public holidays. If animals were in a moribund state, they were sacrificed and necropsied. The examinations of these animals were carried out according to the methods established at the pathology laboratory.
Clinical observations
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal. For technical reasons, however, the clinical observations recorded during the premating period were printed out on a weekly basis. Individual data of daily observations can be found in the raw data.
Food consumption
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter was determined on PND 1 and 4.
Body weight
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals: 1) During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20; 2) females with litter were weighed on the day of parturition (PND 0) and on PND 4.
Females waiting for necropsy were weighed weekly. These body weight data were solely used for the calculations of the dose volume. - Oestrous cyclicity (parental animals):
- The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day. The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.
- Sperm parameters (parental animals):
- For the males, mating and fertility indices (male mating index and male fertility index) were calculated for F1 litters.
- Litter observations:
- Pup number and status at delivery
The status (sex, liveborn or stillborn) and number of all delivered pups were determined as soon as possible on the day of birth. At the same time, the pups were also examined for macroscopically evident changes. Pups that die before this initial examination are defined as stillborn pups.
Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4.
Sex ratio
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle. The sex of the pups was finally confirmed at necropsy.
Clinical observations
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned.
Body weight
The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). ¿Runts¿ were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups. - Postmortem examinations (parental animals):
- Parental animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Special attention was given to the reproductive organs.
The following weights were determined in all animals sacrificed on schedule: anesthetized animals, epididymides, testes, ovaries.
The following organs or tissues of parental animals were fixed in 4% buffered formaldehyde or in modified Davidson¿s solution: all gross lesions, adrenal glands, pituitary gland, testis (fixed in modified Davidson¿s solution), epididymides (fixed in modified Davidson¿s solution), prostate gland, seminal vesicles, coagulation glands, ovaries (fixed in modified Davidson¿s solution), uterus, oviducts, vagina.
The uteri of all cohabited female F0 parental animals will be examined for the presence and number of implantation sites.
The uteri of apparently nonpregnant animals or empty uterus horns will be placed in 10% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations. Then the uteri will be rinsed carefully under running water. When the examinations are completed, the uteri will be transferred to the Pathology Laboratory for further processing. - Postmortem examinations (offspring):
- All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia by means of CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically. All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding.
- Statistics:
- - Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter: simultaneous com-parison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
- Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions.
- Proportions of affected pups per litter with necropsy observations: pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Weight parameters (pathology): Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians. - Reproductive indices:
- Male mating index, male fertility index, female mating index, female fertility index, gestation index, live birth index, postimplantation loss
- Offspring viability indices:
- Pup viability index, sex ratio
- Clinical signs:
- no effects observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- Dose descriptor:
- NOAEL
- Remarks:
- (for systemic toxicity)
- Effect level:
- > 1 000 mg/kg bw/day
- Sex:
- male/female
- Basis for effect level:
- other: No adverse systemic effects were observed up to the highest dose tested
- Dose descriptor:
- NOAEL
- Remarks:
- (for reproductive performance and fertility)
- Effect level:
- > 1 000 mg/kg bw/day
- Sex:
- male/female
- Basis for effect level:
- other: No adverse effects were observed up to the highest dose tested
- Clinical signs:
- no effects observed
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Sexual maturation:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- no effects observed
- Dose descriptor:
- NOAEL
- Remarks:
- (for developmental toxicity)
- Generation:
- F1
- Effect level:
- 300 mg/kg bw/day
- Sex:
- male/female
- Basis for effect level:
- other: Decreased numbers of implants and delivered pups, and an increased postimplantation loss.
- Reproductive effects observed:
- not specified
- Conclusions:
- The NOAEL for developmental toxicity (decreased numbers of implants and delivered pups, and an increased post-implantation loss) is 300 mg/kg bw and for reproductive performance and fertility is 1000 mg/kg bw/day.
- Executive summary:
In a reproduction toxicity screening study triethanolamine (99.5%) in drinking water was administered to male and female Wistar rats, strain Crl:WI(Han) (10animals/sex/dose) orally by gavage,at dose levels of 0, 100, 300, 1000 mg/kg bw/day. Males and females from the same dose group were mated after a 14 days premating period, overnight in a ratio of 1:1. The females were allowed to deliver and rear their pups until day 4 after parturition. Shortly after PND 4 the parental females were sacrificed and examined. Pups were sacrificed on PND 4 and a gross examination was performed. The male animals were sacrificed 36 days after the beginning of the administration, and examined. The results revealed decreased mean number of implantation sites, increased post-implantation and lower average litter size at the highest dose level. No other treatment-related effects were observed.
This study is acceptable and satisfies the guideline requirement for a reproduction toxicity screening study (OECD 421).
- Endpoint:
- three-generation reproductive toxicity
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Well documented peer-reviewed publication.
- Principles of method if other than guideline:
- Na-LAS (chain length distribution C10-14) was fed for 84 days to 4 groups of weanling rats (3 dose levels, plus control), each dose consisting of 50 animals each of both sexes (PO-generation). When the P0 generation was 107-112 days old, 20 females from each dose group were mated with 20 males from the same group and maintained together for 17 days. The first litters of each generation (Fla- and F2a-generation) were sacrificed at 21 days of age. Ten days after the final litter was sacrificed, all females were remated with different males from the same group to obtain the F1b generation. From the Flb-generation, 20 males and females of each group were selected at weaning to continue their respective diets and to be used for further reproduction studies. Reproduction studies on the F1b and F2b generations were started when the rats were 80 to 85 days old, and were continued until the F3b generation was weaned.
- GLP compliance:
- no
- Limit test:
- no
- Species:
- rat
- Strain:
- other: Charles River
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source:
- Age at study initiation: (P) weanling; (F1) 21 days
- Weight at study initiation: (P) Males: average 59.4-59.9 g; Females: average 57.0-57.3 g; (F1) Males: group weight 183.5-214.2 g; Females: group weight 157.8-193.2 g
- Housing: individual wire bottom cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°F): 76 +/- 3
- Humidity (%): 50 +/- 5
- Photoperiod (hrs dark / hrs light): 12/12 hrs
: - Route of administration:
- oral: feed
- Vehicle:
- other: LAS was administered in feed (Purina Laboratory Meal) - no documentation of dilution prior to addition to meal
- Details on mating procedure:
- premating exposure period 84 days
- Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 2 years ( 3 generations)
- Frequency of treatment:
- continuous in feed
- Details on study schedule:
- - F1 parental animals not mated until 80-85 days old
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 107-112 days old - Remarks:
- Doses / Concentrations:
0.02, 0.1, 0.5% (14, 70, 350 mg/kg bw d)
Basis:
actual ingested - No. of animals per sex per dose:
- 50 males and 50 females per group.
- Control animals:
- yes, concurrent no treatment
- Litter observations:
- Deformities and number of pups, average body weights, feed consumption, feed efficiency.
- Postmortem examinations (parental animals):
- Necropsy, body weight, organ to body weight ratios, routine hematology and histology.
- Postmortem examinations (offspring):
- Necropsy, body weight, organ to body weight ratios, routine hematology and histology.
- Reproductive indices:
- fertility, gestation, parturition, neonatal viability, lactation, and post-weaning growth
- Clinical signs:
- no effects observed
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- no effects to body weight were noted in the initial twelve weeks
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- no effects to body weight were noted in the initial twelve weeks
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Description (incidence and severity):
- Test substance intake: no effects to average food consumption were noted in the initial twelve weeks
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- Dose descriptor:
- NOAEL
- Effect level:
- 350 mg/kg bw/day
- Sex:
- male/female
- Basis for effect level:
- other: 0.5%
- Clinical signs:
- not specified
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Sexual maturation:
- not specified
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- no effects observed
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 350 mg/kg bw/day
- Sex:
- male/female
- Basis for effect level:
- other: 0.5%
- Dose descriptor:
- NOAEL
- Generation:
- F2
- Effect level:
- 350 mg/kg bw/day
- Sex:
- male/female
- Basis for effect level:
- other: 0.5%
- Reproductive effects observed:
- not specified
- Conclusions:
- No significant effects on reproduction were observed at the highest concentration tested.
- Executive summary:
Na-LAS was fed for 84 days to 4 groups of weanling rats for two years (three generations). No significant effects were observed at the highest dose tested and the resulting NOAEL for the parental and both offspring generations was 350 mg/kg bw (0.5%)
Referenceopen allclose all
1000 mg/kg bw/d
F0 parental animal:
Yellow discolored urine for male and female parental animals
Statistically significantly decreased body weight gain of the dams during gestation, body weight 8% below control on gestation day 20
Statistically significantly decreased food consumption in parental females during lactation
Statistically significantly decreased sperm head count in the cauda epididymidis of males
Statistically significantly decreased absolute and relative weight of epididymides, cauda epididymidis and prostate in males
Statistically significantly less implantation sites
Statistically significantly increased post-implantation loss
Statistically significantly smaller litters
F1 parental animals:
Yellow discolored urine for male and female parental animals
Statistically significantly decreased body weight gain of the dams during gestation
Decreased food consumption in parental females during lactation
Statistically significantly decreased absolute and relative weight of epididymides and cauda epididymidis in males
Statistically significantly less implantation sites
Statistically significantly increased post-implantation loss
Statistically significantly smaller litters
300 mg/kg bw/d
F0 or F1 parental animals: No test substance-related adverse effects
100 mg/kg bw/d
F0 or F1 parental animals: No test substance-related adverse effects
F1 or F2 pups: No test substance-related adverse effects
300 mg/kg bw/d
F1 or F2 pups: No test substance-related adverse effects
100 mg/kg bw/d
F1 or F2 pups: No test substance-related adverse effects
Test substance stability:
The stability of test substance in rat diet was demonstrated for a period of 32 days at room temperature in a different batch of comparable quality, which was not used for the study. The homogeneity of the mixtures was verified. The concentration control analyses of the samples taken revealed that the values were within a range of 90-110% of the nominal concentration in all analyses at all time points, with the exception of one concentration in the feed of the high-dose group (88%).
Plasma concentrations of 2 -aminoethanol were below 3 mg/kg for all control animals, <3 - 4 mg/kg for the low dose animals, 8 - 11 mg/kg for the mid dose animals and 60 ¿ 81 mg/kg for the high dose animals.
Toxicokinetic data of 2 -aminoethanol (calculated as 2 -aminoethanol hydrochloride) from this two-generation reproduction toxicity study show a dose dependency of the plasma levels of 2 -aminoethanol in the experimental animals and there with prove the bioavailability of 2 -aminoethanol hydrochloride in principle.
Under these conditions, no test substance-related findings from clinical examinations or gross and histopathology were observed, which indicate that the administration of the test compound via the diet adversely affected the fertility or reproductive performance of the F0 or F1 parental animals up to and including a nominal dose of 300 mg/kg bw/d. Estrous cycle data, mating behavior, conception, gestation, parturition, lactation and weaning as well as sperm parameters, sexual organ weights and gross and histopathological findings of these organs (including differential ovarian follicle counts in the F1 females) were comparable between the rats of all test groups.
At the high-dose level (1000 mg/kg bw/d), absolute and relative weights of epididymides and cauda epididymidis were decreased and, in the F0 generation only, the number of homogenization resistant caudal epididymal sperm was slightly, but significantly reduced. However, histomorphological correlates for these findings were missing.
In the high-dose F0 and F1 generation females (1000 mg/kg bw/d), decreased numbers of implants and increased resorption rates resulted in significantly smaller litters, giving evidence for an adverse effect of the test compound on fertility and/or reproductive performance at high doses. It has to be noted that a dose of 1000 mg/kg bw/d also caused beginning systemic toxicity in these females, as was indicated by reduced food consumption and/or body weight gain during gestation/lactation.
All data recorded during gestation and lactation in terms of embryo-/fetal and pup development gave no indications for any developmental toxicity in the F1 and F2 offspring up to a dose level of 1000 mg/kg bw/d. The test substance did not adversely influence pup viability, body weight, sex ratio and sexual maturation.
Thus, under the conditions of the present two-generation reproduction toxicity study, the NOAEL (no observed adverse effect level) for fertility, reproductive performance and systemic toxicity in parental F0 and F1 Wistar rats is 300 mg/kg bw/d.
The NOAEL for pre-and postnatal developmental toxicity in their offspring is 1000 mg/kg bw/d.
Tables
Mean test substance intake (mg/kg bw/d; minimum value / maximum value)
|
Test group 01 |
Test group 02 |
Test group 03 |
F0 males |
94.3 (72.4 / 102.5) |
283.2 (218.4 / 309.4) |
943.3 (716.7 / 1032.6) |
F0 females (premating) |
96.7 (80.5 / 100.7) |
289.6 (241.2 / 304.9) |
964.4 (792.4 / 1017.8) |
F0 females |
|
|
|
* = Days 1¿14 p.p. only
Absolute organ weights (P-generation)
Compared to the controls (= 100%), the following values (in %) were significantly changed (printed in bold):
|
Male animals |
Female animals |
||||
Group |
01 100 mg/kg bw/d |
02 300 mg/kg bw/d |
03 1000 mg/kg bw/d |
01 100 mg/kg bw/d |
02 300 mg/kg bw/d |
03 1000 mg/kg bw/d |
Brain |
99% |
100% |
97%* |
|
|
|
Cauda epididymis |
99% |
102% |
88%** |
|
|
|
Epididymides |
100% |
101% |
92%** |
|
|
|
Prostate |
92% |
99% |
86%** |
|
|
|
Spleen |
|
|
|
105%* |
107% |
97% |
*: p¿0.05; **: p¿0.01
All other mean absolute weight parameters did not show significant differences compared to the control groups.
The decrease of absolute weights of cauda epididymis, epididymides, and prostate in male top-dose animals (1000 mg/kg bw/d) were considered as treatment-related effects.
The decrease of brain weights in top-dose males (1000 mg/kg bw/d) as well as the increase of spleen weights in low-dose females (100 mg/kg bw/d) was considered as incidental and not treatment-related due to a missing dose-response relationship.
Absolute organ weights (F1 generation)
Compared to the controls (= 100%), the following values (in %)were significantly changed (printed in bold):
|
Male animals |
Female animals |
||||
Group |
11 100 mg/kg bw/d |
12 300 mg/kg bw/d |
13 1000 mg/kg bw day |
11 100 mg/kg bw/d |
12 300 mg/kg bw/d |
13 1000 mg/kg bw/d |
Cauda epididymis |
96% |
99% |
88%** |
|
|
|
Epididymides |
100% |
101% |
91%** |
|
|
|
Kidneys |
99% |
106%* |
111%** |
103% |
106%** |
115%** |
Spleen |
99% |
103% |
92%* |
|
|
|
Thyroid glands |
106% |
99% |
109%* |
110% |
118%** |
111%* |
*: p¿0.05; **: p¿0.01
All other mean absolute weight parameters did not show significant differences compared to the control groups.
The decrease of absolute weights of cauda epididymis and epididymides in male top-dose animals (1000 mg/kg bw/d) were considered to be treatment-related.
The increase of absolute kidney weights of male and female animals in mid- (300 mg/kg bw/d) and top-dose (1000 mg/kg bw/d) groups, respectively, was statistically significant. Because no histomorphological correlate was detected, a treatment-related weight increase was less likely.
The decrease of spleen weights in top-dose males as well as the increase of thyroid glands in top-dose males and mid- and top-dose females, respectively, is considered incidental and not treatment-related due to a missing dose-response relationship.
1000 mg/kg bw/day
- Lower mean number of implantation sites (about 20% below control)
- Increased postimplantation loss (19.4%* [*=p¿0.05] vs. 3.7% in control)
- Lower average litter size (about 33% below control).
300 mg/kg bw/day
- No test substance-related adverse effects
100 mg/kg bw/day
- No test substance-related adverse effects
Most high-dose animals and one low-dose animal showed transient salivation for a few minutes immediately after each treatment. This was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. The slightly lower body weight gain of the 1000 mg/kg females during gestation was likely caused by the increased postimplantation loss rather than a systemic toxic effect of the test compound.
Effect on fertility: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 300 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- Read-across to a Klimisch 1 GLP Guideline study
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Effects on developmental toxicity
Description of key information
Once-daily oral gavage administration of Benzenesulfonic acid. 4-C10-sec-alkyl derivs., compds. with triethanolamine of doses up to 500 mg/kg bw/day AI from GD 6 through 20 resulted in substance-related postdose observations of mouth rubbing and paddling behavior - this was not considered to be an adverse effect. Foetal variations of increased incidences of unossified metatarsal and cervical centrum for litters from animals administered 300 or 500 mg/kg/day AI, compared with controls. These findings did not result in a detrimental effect on the maternal animal or the subsequent litter; as such, the no observed adverse effect level (NOAEL) for the maternal animal and subsequent offspring is 500 mg/kg bw/day AI.
Pregnant female mice were exposed to LAS via gavage on days 6 -15 of gestation. Increased mortality was observed at the two highest doses (300 and 600 mg/kg bw/day). These doses also exhibited retarded weight gain and adverse signs in the necropsy. Pregnancy was comparable, however, for all groups. At doses without maternal toxicity, no differences were observed in any parameters. Because of the very wide range between the 2 mg/kg and 300 mg/kg doses, the maternal NOAEL of 2 mg/kg bw/day must be considered very conservative. The NOAEL for teratogenicity was 300 mg/kg bw/day (Palmer et al., 1971)
Pregnant female rats were given LAS orally in distilled water from gestation days 6 to 15 at doses of 0.2, 2, 300, 600 mg/kg bw/day. Some effects such as decreased weight gain and transient diarrhea occurred at the highest dose. Pregnancy rates were comparable at all doses. Litter parameters were not significantly affected at any dose. No significant differences were observed in visceral anomalies or skeletal variants, with the exception of a marginal retardation of sternabral ossification at the highest dose. The NOAEL was set at 300 mg/kg bw/day both for maternal toxicity and teratogenicity (Palmer et al., 1975).
In a reproduction/developmental toxicity screening study with TEA, performed according to OECD guideline 421, Wistar rats 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.At the highest dose tested, 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, the NOAEL for post-natal toxicity in the offspring was 1000 mg/kg bw/day, and the NOAEL for pre-natal developmental toxicity was determined to be 300 mg/kg bw/day (BASF, 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).
The TEA dossier was subject to a
substance evaluation by the UK REACH CA in 2014. In its report
dated August 2015, the UK REACH CA concludes that reproductive toxicity
was not an initial concern for TEA and was not identified as an
additional concern. No further studies or activities were required as
part of the process.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: read-across to Klimisch 1 GLP guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
- Qualifier:
- according to guideline
- Guideline:
- other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Details on test animals or test system and environmental conditions:
- Male and female Wistar rats, strain Crl:WI(Han), supplied by Charles River Laboratories, Research Models and Services, Germany GmbH, which were free from any clinical signs of disease, were used for the investigations. The females were nulliparous and non-pregnant at the beginning of the study. According to a written statement from the breeder, male and female animals were derived from different litters. This was necessary to rule out the possibility of sibling mating. These animals were used as F0 generation parental animals. All other animals used in this study (F1 generation pups) were derived from the supplier-provided animals.
During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
- During overnight matings, male and female mating partners were housed together in Makrolon type M III cages.
- Pregnant animals and their litters were housed together until PND 4.
Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment wooden gnawing blocks (Typ NGM E-022; supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria) were added. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured. The animals were housed in fully air-conditioned rooms in which central air conditioning guaranteed a range of temperature of 20-24 °C and a range of relative humidity of 30-70%. The air change rate was 15 times per hour. There were no or only minimal deviations from these limits. The day/night cycle was 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h.
The animal room was completely disinfected using a disinfector ("AUTEX" fully automatic, formalin-ammonia-based terminal disinfection) before use. Walls and floor were cleaned each week with water containing about 0.5% Incidin Extra N (supplied by Ecolab Deutschland GmbH, Hanau, Germany) and 0.5% Mikro-Quat (supplied by Ecolab GmbH & Co. OHG, Düsseldorf, Germany). The food used was ground Kliba maintenance diet mouse/rat ¿GLP¿ meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, which was available to the animals ad libitum throughout the study (from the day of supply to the day of or the day before necropsy). Drinking water was supplied from water bottles (ad libitum). The bedding used was Lignocel FS 14 fibres, dustfree bedding, supplied by SSNIFF, Soest, Germany.
The 45 male and 45 female rats were 9 weeks old when they arrived from the breeder. During an acclimatization period of about 6 days, animals with lowest and highest body weights were eliminated from the study and used for other purposes. The 40 male and 40 female animals included in the study were 10 weeks old at the beginning of treatment, and their body weights varied between:
- male animals: 357.7 g - 301.4 g
- female animals: 173.2 g - 205.8 g
The assignment of the animals to the different test groups was carried out using a randomization program, according to their weight four days before the beginning of the administration period (day -4). - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- The test substance solutions in drinking water were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification. The maximum period for which each preparation was used was 7 days. For the preparation of the administration solutions the test item was weighed in a graduated measuring flask depending on the dose group, topped up with drinking water and subsequently thoroughly shaken until completely dissolved.
The volume administered each day was 10 ml/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.
Analyses of the test substance preparations
The analyses were carried out at at Competence Center Analytics, BASF SE, Ludwigshafen, Germany. Analytical verifications of the stability of the test substance in drinking water for a period of 7 days at room temperature were carried out prior to the start of the study. Given that Triethanolamin rein is completely miscible with drinking water, solutions were considered to be homogenous without further analysis. Samples of the test substance solutions were sent to the analytical laboratory twice during the study period for verification of the concentrations. Of each sample, one additional reserve sample was retained. Details of the sampling schedule were recorded with the raw data. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The concentration of the test substance in the vehicle (drinking water) was checked by capillary electrophoresis (CE) with internal standard quantification, using a Beckman P/ACE MDQ automated capillary electrophoresis system including capillary oven and UV-detector.
- Details on mating procedure:
- In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group.
The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "GD 0" and the following day "GD 1". - Duration of treatment / exposure:
- Premating period of 2 weeks and a mating period (max. 2 weeks) in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females.
- Frequency of treatment:
- Daily
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Maternal examinations:
- Mortality
A check for moribund or dead animals was made twice daily on working days or once daily on Saturdays, Sundays or public holidays. If animals were in a moribund state, they were sacrificed and necropsied. The examinations of these animals were carried out according to the methods established at the pathology laboratory.
Clinical observations
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal. For technical reasons, however, the clinical observations recorded during the premating period were printed out on a weekly basis. Individual data of daily observations can be found in the raw data.
Food consumption
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter was determined on PND 1 and 4.
Body weight
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals: 1) During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20; 2) females with litter were weighed on the day of parturition (PND 0 ) and on PND 4.
Females waiting for necropsy were weighed weekly. These body weight data were solely used for the calculations of the dose volume.
The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day. The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.
For the males, mating and fertility indices (male mating index and male fertility index) were calculated for F1 litters.
Postmortem examinations
Parental animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Special attention was given to the reproductive organs.
The following weights were determined in all animals sacrificed on schedule: anesthetized animals, epididymides, testes, ovaries.
The following organs or tissues of parental animals were fixed in 4% buffered formaldehyde or in modified Davidson¿s solution: all gross lesions, adrenal glands, pituitary gland, testis (fixed in modified Davidson¿s solution), epididymides (fixed in modified Davidson¿s solution), prostate gland, seminal vesicles, coagulation glands, ovaries (fixed in modified Davidson¿s solution), uterus, oviducts, vagina.
The uteri of all cohabited female F0 parental animals will be examined for the presence and number of implantation sites.
The uteri of apparently nonpregnant animals or empty uterus horns will be placed in 10% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations. Then the uteri will be rinsed carefully under running water. When the examinations are completed, the uteri will be transferred to the Pathology Laboratory for further processing. - Fetal examinations:
- Pup number and status at delivery
The status (sex, liveborn or stillborn) and number of all delivered pups were determined as soon as possible on the day of birth. At the same time, the pups were also examined for macroscopically evident changes. Pups that die before this initial examination are defined as stillborn pups.
Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4.
Sex ratio
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle. The sex of the pups was finally confirmed at necropsy.
Clinical observations
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned.
Body weight
The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). ¿Runts¿ were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.
Postmortem examinations
All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia by means of CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically. All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding. - Statistics:
- - Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter: simultaneous com-parison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
- Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions.
- Proportions of affected pups per litter with necropsy observations: pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Weight parameters (pathology): Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians. - Indices:
- Parental animals: male mating index, male fertility index, female mating index, female fertility index, gestation index, live birth index, postimplantation loss.
Offspring: pup viability index, sex ratio - Details on maternal toxic effects:
- Maternal toxic effects:no effects
Details on maternal toxic effects:
Most high-dose animals and one low-dose animal showed transient salivation for a few minutes immediately after each treatment. This was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. The slightly lower body weight gain of the 1000 mg/kg females during gestation was likely caused by the increased postimplantation loss rather than a systemic toxic effect of the test compound.
No other test substance-related adverse effects were observed. - Dose descriptor:
- NOAEL
- Effect level:
- > 1 000 mg/kg bw/day
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 300 mg/kg bw/day
- Basis for effect level:
- other: developmental toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
The following test substance-related adverse effects/findings were noted:
1000 mg/kg bw/day
- Lower mean number of implantation sites (about 20% below control)
- Increased postimplantation loss (19.4%* [*=p¿0.05] vs. 3.7% in control)
- Lower average litter size (about 33% below control).
300 mg/kg bw/day
- No test substance-related adverse effects
100 mg/kg bw/day
- No test substance-related adverse effects - Dose descriptor:
- NOAEL
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- not specified
- Basis for effect level:
- changes in litter size and weights
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- The NOAEL for developmental toxicity (decreased numbers of implants and delivered pups, and an increased postimplantation loss) is 300 mg/kg bw.
- Executive summary:
In a reproduction toxicity screening study triethanolamine (99.5%) in drinking water was administered to male and female Wistar rats, strain Crl:WI(Han) (10 animals/sex/dose) orallyby gavage,at dose levels of 0, 100, 300, 1000 mg/kg bw/day. Males and females from the same dose group were mated after a 14 days premating period, overnight in a ratio of 1:1. The females were allowed to deliver and rear their pups until day 4 after parturition. Shortly after PND 4 the parental females were sacrificed and examined. Pups were sacrificed on PND 4 and a gross examination was performed. The male animals were sacrificed 36 days after the beginning of the administration, and examined. The results revealed decreased mean number of implantation sites, increased post-implantation and lower average litter size at the highest dose level. No other treatment-related effects were observed.
This study is acceptable and satisfies the guideline requirement for a reproduction toxicity screening study (OECD 421).
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: basic data given
- Principles of method if other than guideline:
- Method: Chernoff-Kavlok teratogenicity screening test
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- mouse
- Strain:
- CD-1
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, New York
- Age at study initiation: 6-8 weeks
- Fasting period before study: none
- Housing: individually house in polycarbonate shoebox cages
- Diet: Purina Certified Rodent Chow 115002 ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1 (72 +/-3°F)
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The daily dosing solutions were prepared and color coded by the chemistry staff at study initiation and were stored refrigerated in amber glass vials to prevent photodegradation. All dosing concentrations were verified as accurate by the chemistry staff. The animal technicians dosing the animals were not aware of the test article name and were blind to doses. Each test article was identified by the number assigned by the Radian Corp. and a corresponding color. This technique allowed for unbiased clinical observations.
VEHICLE
- Amount of vehicle (if gavage): 10 mg/kg bw - Analytical verification of doses or concentrations:
- yes
- Details on mating procedure:
- no data
- Duration of treatment / exposure:
- on days 6-15 of gestation
- Frequency of treatment:
- daily
- Duration of test:
- until post partum day 3
- No. of animals per sex per dose:
- 3 virgin females (Phase I)
2-4 mated females (Phase II)
50 mated females (Phase III) - Control animals:
- yes, concurrent vehicle
- Details on study design:
- Sex: female
Duration of test: up to 3 days post partum - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:
BODY WEIGHT: Yes
- Time schedule for examinations:
Phase 1: at the time of randomization, on day 1, 5 of treatment and day 3 after treatment
Phase 2 : at the time of randomization, on days 6 through 15 and day 17 of gestation,
Phase 3 : at the time of randomization, on days 6 through 15 and day 17 of gestation, post partum day 0 and 3
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 17 (phase 2); Sacrifice on day 3 post partum
- Organs examined: uteri - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes - Fetal examinations:
- - External examinations: No data
- Soft tissue examinations: No data
- Skeletal examinations: No data
- Head examinations: No data - Statistics:
- An overall test for homogeneity of variance (Bartlett's test) and F-test were performed on the weight data of each group following randomization. Average body weight per group and average body weight change per group were calculated for treatment and control groups. Probit analysis of mortality and morbidity data generated in Phase II of the range finding study was used to determine the predicted LD10 for the Phase III.
The following statistical procedures were used for analyzing maternal data:
- Random weights: analysis of variance (ANOVA) (2-tail)
- Survival: Fisher's exact test (one-tail)
- Weight gains: Mann-Whitney U-Test (2-tail)
- Proportion of viable litters Fisher's exact test (one-tail)
- Survival of pups Chi-Square test (one-tail)
The Mann-Whitney U-test (2-tail) was used to compare each group to the concurrent control.
- Number live pups/litter (day 0, day 3)
- Length of gestation
- Average wt. pup (day 0, day 3)
- Average wt. gain/litter (day 3-day 0)
The p-value listed is not corrected for multiple comparisons. - Details on maternal toxic effects:
- Maternal toxic effects:no effects
Details on maternal toxic effects:
See Remarks on results section. - Dose descriptor:
- NOAEL
- Effect level:
- 1 125 mg/kg bw/day
- Basis for effect level:
- other: developmental toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 1 125 mg/kg bw/day
- Basis for effect level:
- other: maternal toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:no effects
Details on embryotoxic / teratogenic effects:
See Remarks on results section. - Dose descriptor:
- NOAEL
- Effect level:
- > 1 125 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- not specified
- Basis for effect level:
- other: Highest dose tested
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Executive summary:
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).
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Acceptable, well-documented publication/study report which meets basic scientific principles.
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- After overnight mating, the rats were randomly allocated to five groups which included one control group and four different treatment groups. LAS was prepared daily as a series of graded aqueous solutions. Animals in all groups were dosed orally at the standard volume of 1.0 mL/100 g. Control animals were dosed in a similar manner with distilled water used as the vehicle. The dams were observed daily for signs of toxicity and weighed on days 1, 3, 6, 10, 14, 17 and 20 of pregnancy. On day 20, the rats were killed by CO2 euthanasia. Their ovaries and uterine contents were examined immediately for number of copora lutea, number of viable young, number of resorption sites, litter weight, and fetal abnormalities.
- GLP compliance:
- no
- Limit test:
- no
- Species:
- rat
- Strain:
- other: Charles River CD strain
- Route of administration:
- other: oral in distilled water
- Vehicle:
- not specified
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- day 6 to day 15 of pregnancy
- Frequency of treatment:
- daily
- Duration of test:
- 20 days
- No. of animals per sex per dose:
- no data
- Control animals:
- yes, concurrent no treatment
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
Parent animals were observed daily. Change in bodyweight was not affected by treatment at 0.2, 2.0, and 300 mg/kg, but treatment at 600 mg/kg was associated with retarded weight gain and a transient diarrhea following initiation of treatment. The pregnancy rate was comparable at all dosages. - Dose descriptor:
- NOAEL
- Effect level:
- 300 other: mg/kg
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
Embryonic and fetal development were assessed by the incidence of major malformations. The incidence of minor visceral anomalies was unaffected by treatment at any dosage. The distribution of skeletal variants were not statistically significant with the exception of a marginal retardation of sternebral ossification at 600 mg/kg. - Dose descriptor:
- NOAEL
- Effect level:
- 300 other: mg/L
- Based on:
- test mat.
- Basis for effect level:
- other: teratogenicity
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- NOAEL = 300 mg/kg for both maternal and teratogenicity
- Executive summary:
Female rats were given LAS orally in distilled water from gestation days 6 to 15 during pregnancy. Some effects such as decreased weight gain and transient diarrhea occurred at the highest dose. Pregnancy rates were comparable at all doses. Litter parameters were not significantly affected at any dose. No significant differences were observed in visceral anomalies or skeletal variants, with the exception of a marginal retardation of sternabral ossification at the highest dose.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Well documented peer-reviewed journal article by researchers at GLP contract testing laboratory.
- Principles of method if other than guideline:
- Twenty female mice were administered 0.2, 2.0, 300 or 600 mg/kg bw of LAS by gavage at days 6-15 of gestation. All animals were sacrificed on day 17 of pregnancy.
- GLP compliance:
- yes
- Remarks:
- not stated, but likely GLP
- Limit test:
- no
- Species:
- mouse
- Strain:
- CD-1
- Details on test animals or test system and environmental conditions:
- Mice were held in plastic containers at standard environmental conditions (20 =/- 1 degrees C, 50 =/- 5% relative humidity) and free access to drinking water and food (Spratt's Laboratory Diet No. 1).
- Route of administration:
- oral: gavage
- Vehicle:
- not specified
- Details on exposure:
- Dosing commenced on day 6 after confirmation of mating by detection of the vaginal plug. Exposure continued until day 15 of gestation.
- Analytical verification of doses or concentrations:
- not specified
- Details on mating procedure:
- Male and female mice were housed five per cage in opaque plastic cages until natural mating occurred.
- Duration of treatment / exposure:
- days 6 - 15 of pregnancy
- Frequency of treatment:
- daily
- Duration of test:
- sacrifice at day 17 of pregnancy
- No. of animals per sex per dose:
- 20 female mice per dose
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- Two doses were chosen to form the basis for safety evaluation (0.2 and 2.0 mg/kg/day) because the likely maximum human intake of detergent from ordinary kitchen use has been estimated at 0.14 mg/kg/day, thus providing factors of 1-2 times the human exposure level. Two further doses were also investigated (300 and 600 mg/kg/day) based on previous toxicity data suggesting that these would impair maternal survival and result in obvious adverse effects.
- Maternal examinations:
- All animals were observed daily for signs of malreaction and were weighed regularly throughout gestation. All animals that died, and survivors at termination, were dissected and examined for macroscopic changes.
- Ovaries and uterine content:
- Ovaries were examined and the number of corpora lutea counted.
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
Among parent animals, treatment at 300 and 600 mg/kg bw d was associated with increased mortality (35% and 90% respectively). At 300 mg/kg bw d weight gain was retarded only during the first four days. No assessment could be made at 600 mg/kg bw d, due to the high mortality rate. Necropsy revealed a ubiquitous occurrence of tympanites, sometimes associated with gastritis. Pregnancy rate was essentially comparable for all groups. - Dose descriptor:
- NOAEL
- Effect level:
- 2 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
At doses with no maternal toxicity, no differences were observed among the dose group and the control group with respect to number of litters, viable young, litter weight, foetal weight, embryonic deaths, implantations and post implantation embryonic loss. At these doses the incidences of major malformations and minor abnormalities were not affected. At the 300 mg/kg bw/day dose, the incidence of total liter loss was 20%; this was attributed to the high maternal toxicity observed at this dose. Among the nine animals with viable young at this dose, mean litter parameters, including litter size and fetal loss, and incidence of major malformations were not statistically different from controls. Minor anomilies, including gross or visceral and skeletal anomalies were increased. At the 600 mg/kg dose, there were no live births. - Dose descriptor:
- NOAEL
- Effect level:
- 300 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- other: teratogenicity
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- Maternal NOAEL = 2 mg/kg bw/day; Teratogenicity NOAEL = 300 mg/kg bw/day
- Executive summary:
Pregnant female mice were exposed to LAS via gavage on days 6 -15 of gestation. Increased mortality was observed at the two highest doses (300 and 600 mg/kg bw/day). These doses also exhibited retarded weight gain and adverse signs in the necropsy. Pregnancy was comparable, however, for all groups. At doses without maternal toxicity, no differences were observed in any parameters. Because of the very wide range between the 2 mg/kg and 300 mg/kg doses, the maternal NOAEL of 2 mg/kg bw/day must be considered very conservative. The NOAEL for teratogenicity was 300 mg/kg bw/day.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 16 April 2020 - 02 November 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- (adopted 25 June 2018)
- Deviations:
- yes
- Remarks:
- 1 animal in Gp 3 (GD 11) was marked as administered 0.00 mL. Thyroid hormone samples were stored incorrectly at -80°C for less than 24 hours. These study deviations neither affected overall interpretation nor compromised the integrity of study findings.
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- Appearance: Yellow / brown liquid
Purity: 51.8% Active Substance
Correction factor: 1.93
Storage conditions: Sealed container, 15 to 25°C, protected from light.
Lot number: 200032320 - Species:
- rat
- Strain:
- other: Crl:WI(Han)
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Margate, United Kingdom
- Age at study initiation: 9 to 10 weeks old
- Weight at study initiation: 140 g
- Fasting period before study: No
- Housing: Animals were housed in cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Acclimation was limited by mated status
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 15 air changes/hour
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was controlled automatically to give a cycle of 12 hours of light and 12 hours of dark.
IN-LIFE DATES: From: To: 20 April 2020 - 11 May 2020 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- Purified
- Analytical verification of doses or concentrations:
- yes
- Details on mating procedure:
- Mating (overnight at the supplier’s laboratory) was confirmed by the presence of a vaginal plug in situ, or other evidence of mating, if necessary. The day on which mating was confirmed was designated as Gestation Day (GD) 0.
- Duration of treatment / exposure:
- From Gestation Day (GD) 6 through 20
- Frequency of treatment:
- Daily
- Duration of test:
- GD 6 to 20
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Control (Purified water)
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 500 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 20 females per dose
- Control animals:
- yes, concurrent vehicle
- Maternal examinations:
- Assessment of toxicity was based on mortality, clinical and postdose observations, body weights, food consumption, and clinical and anatomic pathology of the thyroid. Necropsies were performed on all animals on GD 21 and all macroscopic abnormalities and thyroid weights were recorded for the maternal animals. Microscopic examinations of the thyroid gland were also performed. The progress and outcome of pregnancy were also evaluated.
- Ovaries and uterine content:
- Pregnancy status
Gravid uterus weight
Terminal body weight (recorded for adjusted gravid uterus weight calculations only and have not been reported)
Number of corpora lutea
Number and intrauterine position of implantations subdivided into:
Live fetuses
Early intrauterine deaths
Late intrauterine deaths
Dead fetuses - Fetal examinations:
- Individual fetal and placental weights were recorded; fetuses were examined externally and sexed, and anogenital distance was recorded.
Fetal abnormalities were classified as malformations (rare and/or potentially lethal defects) or variations (commonly occurring non-lethal abnormalities). - Statistics:
- Data for each fetal sex were analyzed separately; only data collected on or after the first day of dosing were analyzed statistically.
Data from non-pregnant animals were excluded from statistical analysis.
The pairwise comparisons of interest were: Groups 2, 3, and 4 versus Group 1.
Analysis of variance (ANOVA) and pairwise comparisons, as applicable, were used to analyze the following.
• Body weight
• Body weight change
• Food consumption
Prior to performing the ANOVA, Levene’s test was performed to test for equality of variances between groups:
All statistical tests were evaluated at the 5.0, 1.0, and 0.1% probability levels. - Historical control data:
- Historical control data included were generated outside the scope of this study; therefore, no claim of compliance will be made for these data. Data were generated from studies performed within the GLP laboratory, by GLP trained staff, whether a claim of GLP compliance was made or not, and were included in the compilation of historical control ranges without bias.
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No substance-related clinical observations were noted at any dose level administered.
Postdose observations during return to home cage consisted of mouth rubbing and excessive salivation and were observed in all test article-treated groups, with the incidence increasing with dose levels. Mouth rubbing was observed 1 hour postdose for one animal administered 500 mg/kg/day AI (Animal R0313). These observations were considered attributable to the unpalatable nature of the test article formulations and did not represent systemic toxicity; as such, these observations were considered nonadverse. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Description (incidence):
- No substance-related unscheduled deaths occurred.
One female administered 500 mg/kg/day AI (Animal R0312) was sacrificed on GD 11. No clinical observations were noted for this animal until GD 11, when decreased activity, excessive salivation, piloerection, audible respiration, and a thin appearance were recorded. Due to the severity of these observations, this animal was subsequently terminated. The animal was confirmed as pregnant at necropsy, and was recorded as thin with a thick caudal region of the esophagus. Although the etiology of this finding was unclear, in isolation, this was considered not substance related. - Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No substance-related effects on body weights or body weight change were noted.
The statistically significant intergroup differences detected were considered to have arisen incidentally and were not associated with the test article. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Substance-related reductions in mean food consumption were observed following the start of administration of 500 mg/kg/day AI compared with controls (GD 6 through 8; P < 0.001). Improvement was observed thereafter, and as such, these initial reductions were considered not to represent an adverse effect of the test article.
No test article-related effect on food consumption was observed following 100 or 300 mg/kg/day AI. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- No substance-related changes in total thyroxine (T4), thyroid stimulating hormone (TSH) or total thyroid triiodothyronine (T3) were observed.
- Endocrine findings:
- no effects observed
- Description (incidence and severity):
- No substance-related changes in total thyroxine (T4), thyroid stimulating hormone (TSH) or total thyroid triiodothyronine (T3) were observed.
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- effects observed, treatment-related
- Description (incidence and severity):
- Paddling behavior was noted for four animals administered 500 mg/kg/day AI (and one female administered 300 mg/kg/day AI (Animal R0204) during return to home cage. These observations were considered attributable to the unpalatable nature of the test article formulations and did not represent systemic toxicity; as such, these observations were considered nonadverse.
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- No test article-associated thyroid weight or thyroid:body weight ratio changes were recorded for adults terminated on GD21.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- All tissues were macroscopically unremarkable or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- Microscopic evaluation was limited to the thyroid gland and macroscopic abnormalities for all animals; no test article-associated changes were recorded for adults terminated on GD21. All tissues were microscopically unremarkable or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age.
- Histopathological findings: neoplastic:
- not examined
- Number of abortions:
- no effects observed
- Description (incidence and severity):
- Of the 20 females in each group, 18, 19, 18, or 19 females administered 0, 100, 300 or 500 mg/kg/day AI, respectively, were pregnant with live fetuses at the scheduled sacrifice. No abortions were reported.
- Pre- and post-implantation loss:
- no effects observed
- Total litter losses by resorption:
- effects observed, non-treatment-related
- Description (incidence and severity):
- One female administered 300 mg/kg/day AI (Animal R0216) had a total in utero litter loss. This was an isolated finding, and in the absence of a dose relationship, was considered to have arisen incidentally.
- Early or late resorptions:
- no effects observed
- Dead fetuses:
- no effects observed
- Changes in pregnancy duration:
- no effects observed
- Changes in number of pregnant:
- no effects observed
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 500 mg/kg bw/day (actual dose received)
- Based on:
- act. ingr.
- Basis for effect level:
- body weight and weight gain
- clinical biochemistry
- clinical signs
- food consumption and compound intake
- gross pathology
- histopathology: non-neoplastic
- mortality
- organ weights and organ / body weight ratios
- Key result
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- not examined
- Reduction in number of live offspring:
- not examined
- Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- no effects observed
- Anogenital distance of all rodent fetuses:
- no effects observed
- Description (incidence and severity):
- No substance-related changes for fetal anogenital distance were observed following maternal administration of the test article. Statistical analysis of the data did not reveal any significant intergroup differences.
- Changes in postnatal survival:
- not examined
- External malformations:
- no effects observed
- Skeletal malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Fetal variations of higher incidences of unossified metatarsals and cervical centrum were observed for litters from animals administered 500 mg/kg/day AI, compared with controls. Higher incidences of unossified cervical centrum were also observed for litters from females administered 300 mg/kg/day AI, compared with controls. There was no overarching pattern of effect observed, and the ossification deficits noted do not impact viability and are most likely transient; therefore, the increased incidence of these variations were considered not to represent an adverse effect of the substance.
- Visceral malformations:
- no effects observed
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 500 mg/kg bw/day (actual dose received)
- Based on:
- act. ingr.
- Sex:
- male/female
- Basis for effect level:
- reduction in number of live offspring
- changes in sex ratio
- changes in litter size and weights
- external malformations
- skeletal malformations
- visceral malformations
- Key result
- Abnormalities:
- no effects observed
- Key result
- Developmental effects observed:
- no
- Lowest effective dose / conc.:
- 500 mg/kg bw/day (actual dose received)
- Treatment related:
- no
- Conclusions:
- The no observed adverse effect level (NOAEL) for the maternal animal and subsequent offspring is 500 mg/kg bw/day AI, based no adverse effects occurring in maternal animals or foetuses.
- Executive summary:
Once-daily oral gavage administration of 0 (control article [vehicle]), 100, 300, or 500 mg/kg bw/day AI of Benzenesulfonic acid. 4-C10-sec-alkyl derivs., compds. with triethanolamine from GD 6 through 20 resulted in substance-related postdose observations of mouth rubbing and paddling behavior. Initial reductions in food consumption were observed for females administered 500 mg/kg bw/day AI, and foetal variations of increased incidences of unossified metatarsal and cervical centrum for litters from animals administered 300 or 500 mg/kg bw/day AI, compared with controls. These findings did not result in a detrimental effect on the maternal animal or the subsequent litter; as such, the no observed adverse effect level (NOAEL) for the maternal animal and subsequent offspring is 500 mg/kg bw/day AI.
Referenceopen allclose all
Phase I: (range finding)
Mortality:
No deaths occurred.
Body weight development:
Dose (mg/kg bw) |
10 |
100 |
1000 |
TD 5 TD 1 |
-0.5 |
+1.2 |
+1.3 |
PTD 7 TD 5 |
+1.9 |
+2.3 |
+0.3 |
PTD 7 TD 1 |
+1.4 |
+3.5 |
+1.6 |
Clinical signs
No signs of systemic toxicity were observed at any dose level.
Phase II: (range finding)
Body weight development
Dose mg/kg bw/day |
600 |
1200 |
2400 |
4800 |
9600 |
TD 10 - TD 1 |
+13.33 |
+12.66 |
+6.55 |
+15.01 |
- |
PTD 2 - TD 10 |
-4.06 |
+7.27 |
+2.97 |
+6.70 |
- |
PTD 2 - TD 1 |
+17.39 |
+19.93 |
+9.52 |
+21.71 |
- |
Mortality and Pregnancy Status
Dose mg/kg bw/day |
600 |
1200 |
2400 |
4800 |
9600 |
No of animals treated |
4 |
4 |
4 |
4 |
4 |
Animals found dead |
1 |
0 |
1 |
2 |
4 |
Ammonium sulfide positive |
1 |
- |
- |
2 |
2 |
Ammonium sulfide negative |
- |
- |
1 |
- |
2 |
Animals sacrificed |
3 |
4 |
3 |
2 |
0 |
Live pups |
3 |
2 |
1 |
2 |
- |
Ammonium Sulfide positive |
- |
- |
1 |
- |
- |
Ammonium sulfide negative |
- |
2 |
1 |
- |
- |
Clinical signs
600 mg/kg
A rough haircoat was seen in all animals. On treatment day 4, one animal was noted as having hunched posture, pale eyes and extremities. This animal was found dead during the morning observation on treatment day 5. This did not appear to be caused by dosing trauma. Two of the surviving animals appeared normal by treatment day 9. One surviving animal exhibited a rough haircoat until post-treatment day 2.
1200 mg/kg
All animals at this dose level exhibited a rough haircoat by treatment day 2. This was the only deviation from normal noted for these animals. All animals appeared normal by treatment day 9.
2400 mg/kg
All animals at this dose level exhibited a rough haircoat by treatment day 2. On post-treatment day 1, one animal appeared languid, exhibited hunched posture, rough haircoat and squinted eyes. This animal was found dead during the morning observation of post-treatment day 2. This death did not appear to be caused by dosing trauma.
4800 mg/kg
Animals at this dose level exhibited a rough haircoat by treatment day 2. On treatment day 4, one animal was noted as not using its left rear leg. This animal¿s leg appeared normal by the afternoon observation on treatment day 5. Two animals were found dead, onde during the morning observation of treatment day 7 and one during the morning observation of treatment day 9. These deaths did not appear to be caused by dosing trauma.
9600 mg/kg
All animals in this dose level died. One animal was found dead during the afternoon observation period of treatment day 1. The remainder of the animals were found dead during the morning observation of treatment day 2. These deaths did not appear to be caused by dosing trauma.
Conclusion
Based on the data obtained from phases I and II, the recommended dose for phae III was 1125 mg/kg/day (LD10).
Phase III
Mortality
There were no deaths in the treatment and control group.
Clinical signs
One animal was noted as having a subcutaneous lump (treatment day 2). This did not appear to be related to treatment. One other animal exhibited hunched posture, languid behavior, rough haircoat,, wheezing, labored respiration, head tilt, squinted eyes, and pale extremities throughout the treatment and post-treatment period. All other animals appeared normal throughout the treatment and post-treatment period.
Maternal body weights
No statistically significant group differences were found for the randomization weights. Statistical analysis was performed to compare the maternal body weights on postpartum day 0 and 3. The treatment group was significantly heavier from the control group at postpartum day 0 but not at postpartum day 3. The average weight gain from pretreatment to postpartum day 0 was significantly greater for the treatment group.
Reproductive index and litter data
No statistically significant effect was observed upon the percent viable litters and upon pup survival.
Duration of gestation
No statistically significant group differences were noted in the length of gestation.
Pup weight data
No statistically significant differences were noted.
Conclusion
Triethanolamine(1125mg/kg/day) did not affect maternal mortality, the number of viable litters, bitter size, percent survival of the pups, birth weight of the pups and weight gainedbythe pups. Using the ranking system of Hardin with a maximum score of 22 and for low maternal toxicity the following scores should be subtracted: viable litters (4); litter size (2); percent survival of pups(3);birth weight of pups(1);and weight gain of pups (2).
Therefore, the score for triethanolamine is 12 which represents intermediate priority for further testing. Since the dose used of triethanolamine did not result in maternal mortality and since the other two ethanolamines (mono- and di-) are positive in the Chernoff/Kavlock test,it is recommended that triethanolamine be retested in the Chernoff/Kavlock test at a higher dose which results in approximately10%maternal mortality. Until it is retested, triethanolamine is judged to be inaccurately tested in the Chernoff/Kavlock preliminary developmental toxicity test.
Pregnancy/litter data: The litter parameters assessed included litter size, fetal loss and litter weight. These parameters were not significantly affected by any dosage. Mean pup weights were statistically higher at 0.2, 2.0 and 300 mg/kg.
The maternal NOAEL of 2 mg/kg bw d is considered very conservative because the range (2-300 mg/kg bw d) was too wide, especially considering the repeated dose toxicity studies (section 7.5.1) which give much higher NOAEL values.
| Variation | Dose Level (AI): | 0 (Control) | 100 mg/kg/day | 300 mg/kg/day | 500 mg/kg/day | Historical control data | |||||||
|
|
44 | 53 | 72 | 78* | 0 to 95% | |||||||
|
14.72 | 16.05 | 21.85 | 33.53 | |||||||||
|
|
0 | 5 | 0 | 17 | 0 to 26% | |||||||
|
0 | 1.05 | 0.00 | 4.23 |
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 500 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- Klimisch 1 GLP guideline study
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
Based on reliable studies conducted on structural analogues and on the substance itself, LAS TEA does not need to be classified for toxicity to reproduction.
Explanation for no classification of TEA:
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) the two generation reproduction toxicity study in Wistar rats with dietary MEA administration demonstrated clear NOAELs for systemic and reproductive toxicity including fertility at 300 mg MEA-HCl/kg bw/day. 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 to 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 to 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 mechanistic in vitro and in vivo studies 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 hepatocytes in 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.
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
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