Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
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:
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 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
The following test substance-related effects/findings were recorded:

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
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
The following test substance-related effects/findings were recorded:

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
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

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
(100 mg/kg bw/d)

Test group 02
(300 mg/kg bw/d)

Test group 03
(1000 mg/kg bw/d)

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
(F1 litter)
- gestation period
- lactation period*



103.5 (92.6 / 111.6)
99.2 (81.6 / 120.2)



315.2 (284.8 / 337.9)
306.7 (249.7 / 370.3)



1043.2 (989.4 / 1084.7)
866.0 (668.6 / 1053.9)

* = 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.

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.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
read-across to 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
Additional information

LAS Na:

No studies on reproduction toxicity are available for LAS Na (10 -13). Data with LAS Na (C10 -14; CAS 69669 -44 -9) were used. LAS Na (C10 -14) 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. Exposure took place during the remating period of 2 weeks and a mating period (max. 2 weeks) in both sexes, approximately 1 week post-mating in males, and during the entire gestation period as well as 4 days of lactation in females. 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).

No two-generation reproduction toxicity study is available for TEA. 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).


Short description of key information:
See below section 'discussion'.

Justification for selection of Effect on fertility via oral route:
Effects were observed on reproduction parameters: decreased absolute and relative weights of epididymides and cauda epididymidis (F0); however, no histopathological correlation.

Effects on developmental toxicity

Description of key information
See below section 'discussion'.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
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:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals 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).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
read-across to Klimisch 1 GLP guideline study
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Quality of whole database:
Information is available only for TEA and not for LAS Na. Therefore, no sufficient data exist in order to assess the potential developmental toxicity of LAS TEA after dermal exposure.
Additional information

Two studies are available addressing the potential developmental toxicity of LAS Na in rats:

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).

Based on the results of the aforementioned studies LAS Na is not considered to be a developmental toxicant.

The following studies were used to address the developmental toxicity requirement for TEA:

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).

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

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

 

In another comparable to guideline prenatal developmental toxicity study (Liberacki, 1996) Pregnant Sprague-Dawley rats were exposed dermally to 0, 10, 25, 75 and 225 mg/kg bw/day of MEA. Rats administered 225 mg MEA/kg bw/day exhibited a treatment-related increased incidence of skin irritation and the body weight gain was significantly decreased during the exposure period. Despite maternal effects observed among dams in the high dose group, reproductive and developmental toxicity parameters among exposed rats were unaffected at all dose levels. The NOAEL for maternal toxicity was set at 75 mg/kg bw/day and the NOAEL for developmental toxicity was set at the highest dose level of 225 mg/kg bw/day. Similarly, pregnant rabbits were exposed via the dermal route to 0, 10, 25, and 75 mg/kg bw/day of MEA. The rabbits in the mid and high dose group exhibited signs of skin irritation, severe at the highest dose level. No treatment-related effects were observed on reproductive and developmental toxicity parameters. The NOAEL for maternal toxicity was set at 10 mg/kg bw/day and the NOAEL for developmental toxicity was set at the highest dose level of 75 mg/kg bw/day.

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 SA 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.


Justification for selection of Effect on developmental toxicity: via oral route:
Lowest NOAEL observed

Justification for classification or non-classification

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

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

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

It is possible that the effects of MEA, DEA and TEA on pre- and post-implantation may be mediated by effects on choline homeostasis (as described above) rather than through a direct embryo toxicity. These effects may be inhibition of cholin-uptake in the liver, subsequent perturbation of choline-homeostasis, with subsequent impairment of C1-metabolism, DNA-methylation, lipid metabolism, and intercellular communication. Choline metabolism is connected to Phosphatidylcholine and Betaine. The latter is reported to be central for the synthesis of SAM (S-Adenosyl-Methionine), a principle methylating agent for biosynthetic pathways and maintenance of critical gene methylation patterns (Stott et al. 2004; Zeisel and Blusztajn, 1994). 

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

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

 References

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

 

OECD SIDS (2009). Diethanolamine.

 

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