Carbonic acid, compound with 2-aminoethanol (1:2)

Endpoint summary

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:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

The target substance carbonic acid, compound with 2-aminoethanol (1:2) is formed from the reaction of 2-aminoethanol and carbon dioxide, at a 2:1 molar ratio. The hypothesis of the analogue approach is that the systemic toxicity of the target substance will be determined by the fact that in aqueous conditions of a mammalian system, it rapidly degrades to 2-aminoethanol, and therefore reading across to 2-aminoethanol provides a reasonable worst-case scenario.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source substance: 2-aminoethanol
Target substance: carbonic acid, compound with 2-aminoethanol (1:2)

For further information on purity and impurities please see the full read across justification attached in Section 13 of IUCLID.

3. ANALOGUE APPROACH JUSTIFICATION

The target substance is expected to break down to form the source substance in mammalian systems; both target and source substances would be expected to have the same mechanism of action and therefore similar systemic toxicity. The target substance is essentially a neutralised form of the source substance. Therefore, the target substance would not be expected to display the topical irritancy and/or corrosive properties of the source substance. However, for systemic effects read across is appropriate as the target substance will degrade rapidly to produce the source substance under physiological conditions.

For a full justification of the read across approach see the read across justification attached in Section 13 IUCLID.

Reason / purpose for cross-reference:

read-across source

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

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

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: no pre-and postnatal developmental toxicity

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no pre-and postnatal developmental toxicity

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:

As indicated in the read across justification document, if ingested, the registered substance is expected to decompose to form monoethanolamine and therefore, it is reasonable to assume that the effects reported in this study are a worst-case for the registered substance.

As such, exposure of rats over 32 d in the diet to monoethanolamine established a NOAEL of 300 mg/kg bw/day based on reduced food consumption, and/or body weight gain, as well as organ weight changes unaccompanied by histological findings.

No pre- and post-natal development toxicity was observed in either F1 and F2 generations, at all doses tested, up to 1000 mg/kg bw/day.

This NOAEL can be directly applied to the registered substance.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Justification for selection of Effect on fertility via oral route:
Reliable study on the analogue substance which is expected to represent a reasonable worst case for the repeat dose inhalation toxicity of the registered substance. Findings at the limit dose (1000 mg/kg bw/day) were: yellow discoloured urine, decreased weight of male accessory sex organs, but with no histopathological change.

Effects on developmental toxicity

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sexually mature, virgin Wistar rats (Chbb :THOM (SPF)) supplied by Karl THOMAE, Biberach an der Riss, Germany,
- Age at study initiation: 60 days
- Weight at study initiation: mean weight approx. 223.7 g
- Fasting period before study: none
- Housing: singly in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm²) ;
- Diet: ground Kliba 343 feed rat/mouse/hamster supplied by Klingentalermuehle AG, Kaiseraugst, Switzerland ad libitum
- Water: tap water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Each day the test substance solutions were freshly prepared shortly before the test substance was administered. For the preparation of the solutions, an appropriate amount of the test substance was weighed in a volumetric flask and subsequently topped up with doubly distilled water and intensively shaken.

VEHICLE
- Concentration in vehicle:
- Amount of vehicle (if gavage): 10 ml/ kg bw.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity of the test substance was proven by visual inspection. The content of active ingredient was 99 .4% before the beginning of the study. The reanalysis of the test substance proved its stability (content: 99 .5%)

Details on mating procedure:

- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: [yes]
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy

Duration of treatment / exposure:

day 6 - 15 of gestation

Frequency of treatment:

daily, once per day

Duration of test:

up to day 21 of gestation

No. of animals per sex per dose:

40 dams per dosing group

Control animals:

yes, concurrent vehicle

Details on study design:

On day 0, the animals were assigned to the different test groups according to a randomization plan. The test substance was administered to the animals orally (by gavage) once a day during the period of major organogenesis (day 6 to day 15 p .c .) always at approx. the same time of day (in the morning). The animals of the control group were treated in the same way with the vehicle (doubly distilled water). The volume administered each day was 10 ml/kg body weight. The calculation of the volume administered was based on the individual body weight determined at the beginning of the administration period (day 6 p.c.). On day 20 p.c., the first 25 animals/group were sacrificed in a randomized order and examined macroscopically. The fetuses were dissected from the uterus and further investigated with different methods. The other animals (15/group) were allowed to litter and rear their pups up to day 21 p.p. (post partum). On day 21 post partum (p.p.) or one of the following days the relevant dams and pups were sacrificed and examined macroscopically.

Maternal examinations:

Clinical examinations
Food consumption
With the exception of day 0 p.c. (all animals) and days 0 p.p. and 21 p.p. (for animals with terminal
sacrifice on day 21 p.p. only), the consumption of food was determined on the same days as was body weight. Food consumption was not determined for the females without litter during the lactation period of the dams used in parallel.

Body weight data
All animals were weighed on days 0, 1, 3, 6, 8, 10, 13, 15, 17 and 20 p.c. Body weights of the animals with
terminal sacrifice on day 21 p.p. were additionally determined on the day of birth and on days 4, 7, 14 and
21 p.p. The body weight change of the animals was calculated from these results. Body weights of the animals without litter were not determined during the lactation period of the dams used in parallel.

Corrected body weight gain (net maternal body weight change)
Furthermore, the corrected body weight gain was calculated for all animals with terminal sacrifice on day 20 p.c. (terminal body weight on day 20 p.c. minus weight of the uterus before it was opened minus body
weight on day 6 p.c.).

Clinical symptoms
All animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited. The nesting, littering, and lactation behavior of the dams with terminal sacrifice day 21 p.p. was generally evaluated in the mornings in connection with the daily clinical inspection of the dams. Only if there were any special findings (e.g., animal could not litter, umbilical cord not cut), these specific findings were documented with the dam concerned.

The littering behavior of the relevant dams was also inspected on weekdays (except holidays) in the afternoons in addition to the evaluations in the mornings. These reevaluation were documented separately, but, as before, findings were only recorded with the dams concerned. Moreover, the duration of gestation, the number of live and dead pups at birth and litter size were recorded for the animals with terminal sacrifice on day 21 p.p. For these animals the fertility and the gestation indices were calculated according to the following formulae :

fertility index = (n pregnant animals/ n mated animals) x 100

gestation index = (n animals with litters/ n pregnant animals) x 100

The values listed in the Summary Tables are group means determined from the fertility/gestation indices of the individual animals.

Mortality
A check was made twice a day on working days or once a day (Saturday, Sunday or on public holidays).

Ovaries and uterine content:

Examinations of the dams at termination
Dams with terminal sacrifice on day 20 p.c.
On day 20 p.c., the dams were sacrificed in randomized order by cervical dislocation and the fetuses dissected from the uterus. After the dams had been sacrificed , they were necropsied and assessed by gross pathology. The uterus and the ovaries were removed and the following data were recorded :

Weight of uterus before it was opened
- Number of corpora lutea
- Number and distribution of implantation sites classified as :
• live fetuses
• dead implantations:
a) early resorptions (only decidual or placental tissues visible or according to Salewski from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy)
b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been
opened)

Furthermore, calculations of conception rate and pre- and postimplantation losses were carried out:
- The conception rate (in %) was calculated according to the following formula :

conception rate = (number of pregnant animals/ number of fertilized animals) x 100

- The preimplantation loss (in % ) was calculated according to the following formula:

((number of corpora lutea - number of implantations)/number of corpora lutea) x 100

- The post implantation loss (in % ) was calculated from the following formula:

((number of implantations - number of live fetuses)/number of implantations) x 100

Dams with terminal sacrifice on day 21 p.p.
On day 21 post partum (p. p. ) the relevant dams were sacrificed by cervical dislocation. After the dams had been sacrificed, the following examinations were carried out:
- gross - pathological examination
- staining of uterus according to Salewski for determination of the number of implantations

Furthermore, calculations of conception rate and postimplantation loss were carried out:
- The conception rate (in %) was calculated according to the following formula :

conception rate = (number of pregnant animals/ number of fertilized animals) x 100

- The post implantation loss (in % ) was calculated from the following formula:

((number of implantations - number of live fetuses)/number of implantations) x 100

Fetal examinations:

Examination of the fetuses:
Examination of the fetuses after dissection from the uterus
At necropsy each fetus was weighed, sexed and examined macroscopically for any external findings. The sex was determined by observing the distance between the anus and the base of the genital tubercle and was later confirmed in all fetuses fixed in Bouin's solution by internal examination. If there were discrepancies between the "external" and the "internal" sex of a fetus, the fetus was finally sexed according to the appearance of its gonads. Furthermore, the viability of the fetuses and the condition
of the placentae, the umbilical cords, the fetal membranes and fluids were examined. Individual placental
weights were recorded. After these examinations, approximately one half of the fetuses per dam was placed in ethyl alcohol and the other half was placed in Bouin's solution for fixation and further evaluation.

Soft tissue examination of the fetuses
After fixation in Bouin's solution, approximately one half of the fetuses of the dams of all groups was examined for any findings in the organs according to the method of Barrow and Taylor with special attention being paid to the kidneys and the ureters. After the examination, these fetuses were discarded with the exception of the kidneys, which were placed into cassettes separately for each fetus and kept in 4% formaldehyde solution for possible further examination by light microscopy. Moreover, after fixation of the fetuses placed in ethyl alcohol for further evaluation of the fetal skeletons the organs of these fetuses
were examined macroscopically. Thereafter, the kidneys of each fetus were placed into cassettes and kept in 4% formaldehyde solution for a possible further examination by light microscopy, while the other organs were discarded. Afterwards the carcasses of these fetuses were stained according to a modified method (Dawson) for the presentation of the skeletons.

Skeletal examination of the fetuses
After fixation in ethyl alcohol and examination of the organs, the skeletons of the fetuses were stained according to a modified method of Dawson. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After these examinations the relevant fetuses were retained by litter.

Evaluation criteria for assessing skeletons and organs of the fetuses
In the present investigations the following terms (definitions) were used for describing a change:
- Malformations (concerning external, soft tissue and skeletal observations)
Rare and/or probably lethal changes were classified as malformations (e.g. exencephaly, atresia ani, hernia umbilicalis).

- Variations (concerning external, soft tissue and skeletal observations)
Changes which occur regularly also in control groups and have generally no adverse effect on survival were regarded as variations (e.g. dilated renal pelvis).

- Retardations (concerning skeletal observations only)
Delays in skeletal development compared with the norm at the time of the examination were considered to be retardations (e.g. sternebra(e) not ossified)

- Unclassified observations (concerning external and soft tissue observations, only)
External or soft tissue observations, which could not be classified as malformations or variations (e.g.
blood coagulum around placenta).

Examination of the pups:
Pup number and status at delivery
All pups derived from the females were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn members of each litter. Pups which died before the first determination of their status on the day of birth were designated 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. Dead pups were evaluated by the methods which will be described in detail before.
The number and percentage of dead pups on the day of birth (day 0) and of pups dying between days 1-4, 5-7, 8-14 and 15-21 of the lactation period were determined; however, pups which died accidentally were
not included in these calculations. The number of live pups/litter was calculated on the day of birth, and on
lactation days 4, 7, 14 and 21. Furthermore, viability and lactation indices were calculated according to the
following formulas :

Viability index (%) = (number of live pups on day 4 after birth/ number of liveborn pups on the day of birth)
x 100

Lactation index (%) = (number of live pups on day 21 after birth/ number of live pups on day 4 after birth)
x 100

Sex ratio
On the day of birth (day 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. During the following time the sex of the pups was assessed by the external appearance of the anogenital region and/or the mammary line of the animals and was finally confirmed at necropsy. The sex ratio was calculated for day 0 and day 21 after birth according to the following formula:

sex ratio = (number of live male or female pups on day 0/21 / number of live male and female pups on day 0/21) x 100

Pup body weight data
The pups were weighed on the day after birth (day 1 p.p.) and on days 4, 7, 14 and 21 after birth.
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). In the relevant summary tables pup body weights and pup body weight gains are listed for males, females and males + females.

Pup clinical observations
The pups were examined each day for clinical symptoms (including gross-morphological findings).

Pup necropsy observations
After sacrifice on day 21 p.p. or one of the following days (by means of CO2) or intercurrent death, the pups were examined externally, eviscerated and their organs were assessed macroscopically with special attention being paid to the urinary tract. After the macroscopic examination of the pups, the kidneys of each pup were placed into cassettes and fixed in 4% formaldehyde solution for a possible further examination by light microscopy. If there were notable findings or if abnormalities were found in the daily clinical observation of the animals after their delivery, the affected animals were, if it was deemed necessary, examined additionally using appropriate methods (e.g., skeletal staining according to modified Dawson's method and/or further processing of head according to Wilson's method. The stained skeletons were evaluated under a stereomicroscope or a magnifying glass. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation (with the exception of the kidneys (see above)).

Statistics:

Dunnett-Test was used for a simultaneous comparison of several dose groups with the control. The hypothesis of equal means was tested. This test was performed two-sided and was used for the statistical evaluation of food consumption, body weights and body weight change (females and pups), corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, number of corpora lutea, number of implantations, number of resorptions and number of live fetuses; proportion of preimplantation loss, postimplantation loss, resorptions and live fetuses in each litter; litter mean fetal body weight and litter mean placental weight, duration of gestation and number of pups delivered per litter. For the body weight and the body weight change of the pups the mean weight of each litter was used for the statistical analysis (statistical unit = litter).
Fisher' s Exact Test was used for a pairwise comparison of each dose group with the control for the hypothesis of equal proportions. This test was performed one - sided was used for statistical evaluation of the following parameters: female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings, female fertility index, gestation index, females with liveborn, stillborn and with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy.
The Wilcoxon Test was used for a comparison of each dose group with the control for the hypothesis of equal medians. This test was performed one-sided and was used for the proportion of fetuses with malformations, variations, retardations and/or unclassified observations in each litter and for the proportion of affected pups per litter with necropsy observations. If the results of these tests were significant, labels (*for ≤ 5 0.05, ** for p ≤ 0.01) were printed in the summary tables.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Monoethanolamine caused some signs of maternal toxicity when administered by gavage to pregnant Wistar rats from days 6 - 15 of gestation at the highest dose level tested, 450 mg/kg/day. Maternal toxicity was substantiated by reduced food consumption, lower mean body weights and impaired body weight gain. The oral administration of monoethanolamine at 450 mg/kg/day or doses below this had no influence on resorption rate, number of live fetuses or pups/dam, mean fetal weight or pup body weights.

Dose descriptor:

NOAEL

Effect level:

120 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:
No signs of developmental toxicity occurred up to and including the highest dose level (450 mg/kg/day), especially no substance-induced teratogenic effects were observed neither in the fetuses nor in the pups. Furthermore, there were no indications for any substance-related growth retardations. The urinary tract of the rat fetuses/pups did not show any treatment-related findings. Dilated renal pelvis and/or hydroureter were found in a considerable, but according to historical control data, not unexpected high number of fetuses of all groups including the controls without any relation to dosing, but did not occur at an increased rate in the pups of the substance-related groups. All skeletal malformations, variations, or retardations which occurred did not show a clear dose-response relationship, can be found at comparable or even higher rates within the historical control and/or the differences between the groups are without biological significance.

Dose descriptor:

NOAEL

Effect level:

>= 450 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Results summary:

The following findings were obtained and assessed as substance-related:

Test group 3 (450 mg/kg body weight/day):

- statistically significantly reduced food consumption at the beginning of the treatment period (days 6 -

8 p.c.), the final days of the gestation period (days 17 - 20 p.c.) and during the first days of the

lactation period (days 0- 4 p.p.).

- statistically significantly lower mean dam body weights than the controls on days 15, 17 and 20 p.c. and on days 0, 4, 7 and 21 p.p. ; impaired body weight gain of the dams during posttreatment days 15 - 20 p.c.

 

Test group 2 (120 mg/kg body weight/day) :

- no substance-related effects on dams, fetuses or pups

 

Test group 1 (40 mg/kg body weight/day) :

- no substance-related effects on dams , fetuses or pups

 

Thus, under the conditions of this study, Monoethanolamine pure caused some signs of maternal toxicity when administered by gavage to pregnant Wistar rats from days 6 - 15 p.c. at the highest dose level tested (450 mg/kg body weight/day). Maternal toxicity was substantiated in this dose group by a reduced food consumption, lower mean body weights and impaired body weight gain. 120 and 40 mg/kg body weight did not induce any signs of maternal toxicity in the rats.

There occurred no signs of developmental toxicity up to and including the highest dose level (450 mg/kg body weight/day). The reproductive parameters were unaffected and neither the fetuses nor the pups

showed an increased malformation rate or any indications for a substance-induced growth retardation; especially, the urinary tract of the rat fetuses/pups did not show any treatment-related findings.

Based on these study results, the no observable adverse effect level (NOAEL) on the maternal organism is 120 mg/kg body weight/day and 450 m g/kg body weight/day for the progeny.

 

 

Detailed results:

 

Maternal data:

 

Duration of Pregnancy:

The mean duration of gestation for the 21 postpartum (p.p.) females was 21.8 days, 21.6 days, 21.6 days, and 21.4 days for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. In the females euthanized on day 21 p.p., the duration of gestation and the gestation index were substantially similar in all groups.

Body weight:

The mean maternal body weight of the 450 mg/kg/day group was statistically significantly lower than that of the control group on days 15, 17, and 20 of gestation. The high dose females gained statistically significantly less weight than the controls during the treatment-free interval of the gestation period (days 15-20) and on days 0, 4, 7, and 21 p.p. The results of the corrected body weight gain on gestation day 20 of all groups did not show any differences of biological significance.

Food/water consumption:

The food consumption of the high dose animals (450 mg/kg/day) was statistically decreased within the first days of the treatment period (days 6-8 of gestation) and also after termination of the treatment on the last days of the gestation period (17-20 of gestation). During the beginning of the lactation period (days 0-4 p.p.) there was also a slight, but statistically significant reduction in the food consumption of the high dose animals, Description, severity, time of onset and duration of clinical signs: No signs that might be attributed to the test substance administered were detected during gestation and lactation periods. During gestation, piloerection was recorded for one high dose animal on day 13. Without any dose-response relationship insufficient nesting activity was observed for several dams of all groups. During lactation one dam of the 40 mg/kg/day group was found dead on day 0 p.p. after an incomplete delivery. Moreover one dam of the 120 mg/kg/day group had a total litter loss on day 1 after birth. All of these findings are spontaneous in nature and cannot be attributed to the test substance administration.

Gross pathology incidence and severity:

There were no substance-related observations at necropsy in any of the dams. Hydrometra (a spontaneous finding) was recorded for one female of the control group, for 2 females of the 40 mg/kg/day group, and 3 females of the 120 mg/kg/day group.
These animals did not become pregnant. Edema of the lungs which has to be related to the termination of the rats was recorded for several dams of the control, low and intermediate groups without any relation to dosing. For the one low dose female that died intercurrently during parturition, undelivered pups were found in the uterus. Organ weight changes, particularly effects on total uterine weight: The uterus weights, which were determined for the animals with termination on day 20 of gestation only, were not influenced by the administration of the test substance. The differences between the groups is without biological relevance and do not show any dose-response relationship.

 Fetal data:

Litter size and weights

Of the females euthanized on day 21 p.p. the number of females with liveborn was 12, 10, 13, and 10 and the number of pups delivered was 165, 132, 170, and 125 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. The litter size was not influenced by the test substance administration. The mean fetal body weights were not influenced by the test substance administration. The mean body weight of viable fetuses was 3.9 grams for all groups.

Number viable (number alive and number dead)

Dams with viable fetuses was 21, 20, 20 and 24 and the number of fetuses alive/dead were 293/0, 282/0, 263/0 and 311/0 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively.

Sex distribution and ratio

The sex distribution of the fetuses in the test groups was comparable with the control fetuses. Sex ratios (M/F in %) on day 0 were:

 

control: 54.6/45.4

40 mg/kg/day: 55.7/44.3

120 mg/kg/day: 46.1/53.9

450 mg/kg/day: 53.6/46.4

Sex ratios (M/F in %) on day 21 were:

control: 54.4/45.6

40 mg/kg/day: 54.0/46.0

120 mg/kg/day: 45.2/54.8

450 mg/kg/day: 54.2/45.8

The sex distribution and sex ratios of live pups on the day of birth and on day 21 p.p. did not show any substantial difference between controls and treated test groups.

Organ weights

The mean placental weights in the test groups were not influenced by the test substance administration.

 

 

Grossly visible abnormalities, external, soft tissue and skeletal abnormalities

The only external malformation which was found was an anasarca in one high-dose fetus. This malformation is also present at a low incidence in the historical control data. The external examination of the fetuses revealed no variations in any group. One unclassifed observation, fused placentae, was recorded in one fetus of the 40 mg/kg/day group and one fetus of the 450 mg/kg/day group. In all groups, including the control, some soft tissue malformations were found. These malformations were related to the eyes (microphthalmia), the heart (dilatation of the right or both ventricles; dextrocardia), the lung (uni-lobular) or the kidneys (hyper-/hypoplasia) and did not show any relation to dose. Two soft tissue variations, which were related to the urinary tract (dilated renal pelvis; hydroureter) occurred in all groups without any dose-response relationship and were fully within the historical control range. One unclassified observation (bloody inhibition of the kidneys) was recorded for 3 control and one high dose fetus.

Mortality and day of death

One dam of the 40 mg/kg/day group died intercurrently during delivery. Undelivered pups were found in the uterus.

 

Number pregnant per dose level

The conception rate varied between 85% (450 mg/kg/day group) and 75% (40 mg/kg/day group). The conception per dose level was 33 in the control group, 30 in the 40 mg/kg/day group, 33 in the 120 mg/kg/day group and 34 in the 450 mg/kg/day group. The number pregnant at caesarian-section was 21 in the control, 20 in the 40 and 120 mg/kg/day groups and 24 in the 450 mg/kg/day group. The females euthanized on day 21 p.p. showed no substance-associated effects on the fertility index which was 80%, 67%, 87% and 67% for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively.

Number aborting:

No fetuses were aborted or delivered early in any of the groups.

Number resorptions, early/late if available

Mean early resorptions were 1.4, 0.9, 1.0 and 0.9 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. Mean late resorptions were 0.2, 0.3, 0.1 and 0.0 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. In the animals euthanized on day 20 of gestation, there were no-substance related and/or statistically significant differences in the number of resorptions.

Number of implantations

The mean number of implantation sites for the 20 females euthanized on day 20 of gestation were 15.6, 15.3, 14.3 and 13.8 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. The mean number of implantation sites for the 21 p.p. females were 14.6, 14.6, 14.1 and 13.4 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. In the animals euthanized on day 20 of gestation and the females euthanized on day 21 p.p., there were no-substance related and/or statistically significant differences in the mean number of implantation sites.

Pre and post implantation loss

The mean % pre-implantation loss was 3.4, 6.8, 9.9 and 11.7 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. The mean % post-implantation loss for the 20 gestational females was 10.3, 7.3, 7.0 and 6.3 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. The mean % post-implantation loss for the 21 p.p. females was 5.7, 3.9, 9.7 and 7.3 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. In the animals euthanized on day 20 of gestation, there were no-substance related and/or statistically significant differences in the values calculated for the pre- and postimplantation losses. The females euthanized on day 21 p.p. showed no substance-associated effects on the postimplantation loss.

Number of corpora lutea

The mean numbers of corpora lutea were 16.1, 16.3, 15.6 and 15.7 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. In the animals euthanized on day 20 of gestation, there were no-substance related and/or statistically significant differences in the mean number of corpora lutea.

Postnatal growth

Without any clear relation to dosing, pup weights were occasionally statistically significantly lower in the substance-treated groups than in the respective control values. On day 21 p.p. control and high dose pup weights were substantially similar, whereas the mean pup body weights of the 40 and 120 mg/kg/day group were still slightly, but not significantly lower than the control values. On days 1-4 p.p. pup body
weight gains were also statistically significantly lower in the substance-treated groups, again without a clear dose-response relationship. Because treatment of the dams took place only until day 15 of gestation and because of no clear dose-response relationship, it seems very unlikely that the differences in pup body weight/body weight gain are substance-related.

Postnatal survival

The mean number of delivered pups/dam was not influenced by the administration of test substance. There were no substantial biological relevant differences concerning pup viability/ mortality in any of the groups. Viability and lactation indices were unaffected.

Pup clinical observations

None of the pups of the different groups showed any clinical signs until termination.

Pup necropsy observations

Only a few pups showed findings at necropsy. Post mortem autolysis, incisor sloped and dilated renal pelvis (1 high-dose pup) occurred in single pups of the control, the 40 and 450 mg/kg/day groups.

The only skeletal malformations which occurred were related to the thoracic part of the vertebral column (thoracic vertebral body/bodies dumbbell-shaped (asymmetrical) or bipartite (asymmetrical)). One or both of these malformations were found in a few fetuses of each test group including the controls without any biological relevant differences. The variations elicited were related to the ribs (shortened 13th rib(s), accessory 14th rib(s), rudimentary cervical rib(s), and the sternum (sternebra(e) of irregular shape or bipartite). These variations had no clear dose-response relationship, can be found in a similar frequency in historical control data, and/or the differences between groups are without biological significance. In all groups signs of retardations (incomplete or missing ossification of vertebral bodies/arches and the sternebra(e)) were found. The differences between the groups, however, are not associated with the test substance administration. All of the skeletal retardations are to be found at a comparable frequency in the historical control data and most often a clear dose-response relationship is not present. The only statistically significant difference, an increased rate of total variations in the 120 mg/kg/day group, is without biological relevance because it shows no dose-dependence.

Conclusions:

As indicated in the read across justification document, if ingested, the registered substance is expected to decompose to form monoethanolamine and therefore, it is reasonable to assume that the effects reported in this study are a worst-case for the registered substance.

No effects were reported at the doses tested, with the highest dose tested giving giving a NOAEL of 450 mg/kg bw/day.

This NOAEL can be directly applied to the registered substance.

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

The target substance carbonic acid, compound with 2-aminoethanol (1:2) is formed from the reaction of 2-aminoethanol and carbon dioxide, at a 2:1 molar ratio. The hypothesis of the analogue approach is that the systemic toxicity of the target substance will be determined by the fact that in aqueous conditions of a mammalian system, it rapidly degrades to 2-aminoethanol, and therefore reading across to 2-aminoethanol provides a reasonable worst-case scenario.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source substance: 2-aminoethanol
Target substance: carbonic acid, compound with 2-aminoethanol (1:2)

For further information on purity and impurities please see the full read across justification attached in Section 13 of IUCLID.

3. ANALOGUE APPROACH JUSTIFICATION

The target substance is expected to break down to form the source substance in mammalian systems; both target and source substances would be expected to have the same mechanism of action and therefore similar systemic toxicity. The target substance is essentially a neutralised form of the source substance. Therefore, the target substance would not be expected to display the topical irritancy and/or corrosive properties of the source substance. However, for systemic effects read across is appropriate as the target substance will degrade rapidly to produce the source substance under physiological conditions.

For a full justification of the read across approach see the read across justification attached in Section 13 IUCLID.

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

Principles of method if other than guideline:

Developmental toxicity study conducted on rabbits, study design similar to OECD 416

GLP compliance:

no

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Research Products, Inc. (Denver, PA)
- Weight at study initiation: 3.0 - 4.0 kg
- Housing: Wire bottom cages
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20
- Humidity (%): 40 - 60%
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

dermal

Vehicle:

water

Remarks:

Milli-Q

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of MEA in the dosing solutions, stability throughout the dosing period and homogeneity of the dosing solutions were confirmed analytically.

Details on mating procedure:

- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 2/1
- Proof of pregnancy: The day on which copulation was observed was considered gestation Day 0.

Duration of treatment / exposure:

Days 6 - 18 of gestation

Frequency of treatment:

Daily - 6 hours per day

Duration of test:

Observations were conducted up to Day 29.

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

25 mg/kg bw/day

Dose / conc.:

75 mg/kg bw/day

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results of dermal range-finding and teratology probe studies conducted in rabbits.
- Rationale for animal assignment (if not random): Random assignment

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days 0, 6, 9, 12, 15, 18, 24 and 29

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: Liver, kidneys, gravid uteri

Ovaries and uterine content:

The ovaries and uterine content were examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

Continuous data were evaluated for homogeneity of variance using Levene’s test. Based upon the outcome of these tests, a parametric or non-parametric analysis of variance (ANOVA) was performed. If the ANOVA was significant, analysis by Dunnett’s test, the Wilcoxen Rank-Sum test with Bonferroni correction, or a pooled t-test was performed as appropriate. Level of statistical significance set at α = 0.05.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

Rabbits administered 75 mg/kg/day of MEA exhibited severe skin irritation (erythema, edema, ecchymosis, necrosis, exfoliation and crusting) at the site of exposure. Subsequent to the dosing period, exfoliation, crusting and areas of necrosis persisted. The skin of the majority of these rabbits began to heal as evidenced by scab formation late in the gestation period. Crusting, transient erythema and edema were noted in a few rabbits administered 25 mg/kg/day. No significant dermal irritation or lesions were observed among rabbits administered 10 mg/kg/day.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Although no statistically identified bodyweight changes were observed, the average bodyweight gain of high dose rabbits over the course of gestation was decreased when compared to that of the control and other dose groups, mainly due to weight loss or very little weight gain during the treatment period.

Food consumption and compound intake (if feeding study):

no effects observed

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:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

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

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day

Basis for effect level:

dermal irritation

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically or biologically significant treatment-related differences in the incidence of any fetal variation or malformation, or in the number of malformed fetuses in any dose group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically or biologically significant treatment-related differences in the incidence of any fetal variation or malformation, or in the number of malformed fetuses in any dose group.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically or biologically significant treatment-related differences in the incidence of any fetal variation or malformation, or in the number of malformed fetuses in any dose group.

Other effects:

no effects observed

Dose descriptor:

NOEL

Effect level:

75 mg/kg bw/day

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

skeletal malformations

visceral malformations

Developmental effects observed:

no

Conclusions:

No evidence of developmental or fetal toxicity was observed following dermal exposure of pregnant rabbits to maternally toxic doses of monoethanolamine. Maternal toxicity was evidenced primarily as skin irritation at doses of 25 and 75 mg/kg/bw/day of monoethanolamine.

Executive summary:

This study was conducted on pregnant New Zealand White rabbits and involved the dermal exposure of monoethanolamine to assess developmental toxicity. Doses of monoethanolamine were applied to shaved skin on the back of the rabbits for approximately 6 hr/day on days 6 through 18 of gestation. This period of gestation includes the major period for organogenesis. 2 mL/kg was applied at doses of 0 (control), 10, 25 and 75 mg/kg/day. Clinical observations, bodyweight and food consumption were monitored throughout the study. Once sacrificed on day 29 of gestation, post mortem examinations took place such as gross macroscopic examination and organ weight analysis. The ovaries and uterine content were examined for gravid uterus weight, number of corpora lutea, number of implantations, number of early resorptions and number of late resorptions. Foetal examinations also took place, where the foetuses were examined for external abnormalities, soft tissue abnormalities and skeletal abnormalities. The study reported no maternal toxicity or embryotoxicity. However, rabbits administered with 75 mg/kg/day of monoethanolamine experienced severe skin irritation at the site of exposure. The maternal toxicity NOAEL was determined as 10 mg/kg/day due to the severe skin irritation in the 25 and 75 mg/kg/day treatment groups. The NOAEL for embryo and foetal toxicity was determined as > 75 mg/kg/day as no foetal toxicity was observed..

Reference 3

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

The target substance carbonic acid, compound with 2-aminoethanol (1:2) is formed from the reaction of 2-aminoethanol and carbon dioxide, at a 2:1 molar ratio. The hypothesis of the analogue approach is that the systemic toxicity of the target substance will be determined by the fact that in aqueous conditions of a mammalian system, it rapidly degrades to 2-aminoethanol, and therefore reading across to 2-aminoethanol provides a reasonable worst-case scenario.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source substance: 2-aminoethanol
Target substance: carbonic acid, compound with 2-aminoethanol (1:2)

For further information on purity and impurities please see the full read across justification attached in Section 13 of IUCLID.

3. ANALOGUE APPROACH JUSTIFICATION

The target substance is expected to break down to form the source substance in mammalian systems; both target and source substances would be expected to have the same mechanism of action and therefore similar systemic toxicity. The target substance is essentially a neutralised form of the source substance. Therefore, the target substance would not be expected to display the topical irritancy and/or corrosive properties of the source substance. However, for systemic effects read across is appropriate as the target substance will degrade rapidly to produce the source substance under physiological conditions.

For a full justification of the read across approach see the read across justification attached in Section 13 IUCLID.

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

Principles of method if other than guideline:

Developmental toxicity study conducted on rats, study design similar to OECD 416

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: VAF/Plus CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Weight at study initiation: 250 - 300 g
- Housing: Wire bottom cages
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C
- Humidity (%): 40 - 60 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

dermal

Vehicle:

water

Remarks:

Deionised water

Details on exposure:

TEST SITE
- Area of exposure: Applied directly to backs (clipped free of hair)
- Type of wrap if used: Absorbent gauze pad, followed by non-absorbent cotton and elastic bandage.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Washing with water dampened towel
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1 ml/kg
- Concentration (if solution): 1 - 22.5 %
- Constant volume or concentration used: no - adjusted for bodyweights

USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of monoethanolamine in the dosing solutions, stability throughout the dosing period and homogeneity of the dosing solutions were confirmed analytically.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: Overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Days 6 to 15 of gestation (10 days)

Frequency of treatment:

Daily - 6 hours per day

Duration of test:

Observations were conducted up to Day 21.

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

25 mg/kg bw/day

Dose / conc.:

75 mg/kg bw/day

Dose / conc.:

225 mg/kg bw/day

No. of animals per sex per dose:

30 - 45

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based upon results of dermal range-finding and teratology probe studies in rats.
- Rationale for animal assignment (if not random): Random assignment.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days 0, 3, 6, 16 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: Liver, kidneys and gravid uteri

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

Continuous data were evaluated for homogeneity of variance using Bartlett’s Test. Based upon the outcome, a parametric or non-parametric analysis of variance (ANOVA) was performed. If the ANOVA was significant, analysis by Dunnett’s test, the Wilcoxen Rank-Sum test with Bonferroni’s correction, or a pooled t test was performed as appropriate. Level of statistical significance – α = 0.05.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

Rats administered 225 mg/kg/day exhibited a treatment-related increased incidence of skin irritation at the site of exposure. In general, the dermal irritation followed a progression, beginning with erythema and leading to necrosis, scabs and scar formation. No significant dermal irritation or lesions were observed among rats administered lower doses of monoethanolamine.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The bodyweight gain of rats given 225 mg/kg/day was significantly decreased during the exposure period. No effect on weight gain was observed in dams treated with lower levels of monoethanolamine.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

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

Dose descriptor:

NOEL

Effect level:

75 mg/kg bw/day

Basis for effect level:

body weight and weight gain

dermal irritation

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Dose descriptor:

NOEL

Effect level:

225 mg/kg bw/day

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

skeletal malformations

visceral malformations

Key result

Developmental effects observed:

no

Conclusions:

No evidence of developmental or foetal toxicity was observed following dermal exposure of pregnant rats to maternally toxic doses of monoethanolamine. Maternal toxicity was evidenced primarily as skin irritation and reduced weight gain at the 225 mg/kg/day dose level.

Executive summary:

This study was conducted with rats and involved the dermal exposure of monoethanolamine to assess developmental toxicity. Doses of monoethanolamine were applied to shaved skin on the back of rats for approximately 6 hr/day on days 6 through 15 of gestation. This period of gestation included the major period of organogenesis. 1 mL/kg was applied at doses of 0 (control), 10, 25, 75 and 225 mg/kg/day, with deionised water as the vehicle. Clinical observations, bodyweight and food consumption were monitored throughout the study. Once sacrificed on day 21 of gestation, post mortem examinations took place such as gross macroscopic examination and organ weight analysis. The ovaries and uterine content were examined for gravid uterus weight, number of corpora lutea, number of implantations, number of early resorptions and late resorptions. Foetal examinations also took place, where foetuses were examined for external abnormalities, soft tissue abnormalities and skeletal abnormalities. The study reported very slight maternal toxicity at the 225 mg/kg/day dose level and no embryotoxicity. Maternal toxicity was evidenced as dermal irritation and reduced bodyweight gain. The maternal toxicity NOEL was determined as 75 mg/kg/day, and the NOEL for embryo and foetal toxicity was determined as > 225 mg/kg/day as no foetal toxicity was observed.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

450 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

Justification for selection of Effect on developmental toxicity: via oral route:
Reliable study on the analogue substance which is expected to represent a reasonable worst case for the pre-natal developmental toxicity by the oral route of the registered substance. No signs of developmental toxicity observed up to and including the highest dose level. There were no indications of any substance-related growth retardation.

Justification for selection of Effect on developmental toxicity: via dermal route:
Reliable studies on the analogue substance which is expected to represent a reasonable worst case for the pre-natal developmental toxicity by the dermal route of the registered substance. No signs of developmental toxicity observed up to and including the highest dose level. There were no indications of any substance-related growth retardation. Maternal toxicity was observed in the form of dermal irritation at the highest doses, however this is due to the topical irritancy of monoethanolamine (analogue substance) and would not be expected on dermal exposure to the target substance. Skin irritation studies on the target substance prove it is a non-irritant.

Justification for classification or non-classification

As detailed in the read across justification document, it is expected that data on the analogue substance (monoethanolamine) represents a reasonable worst-case for the registered (target) substance. As such, the available data support non-classification of the registered substance as toxic to reproduction, according to the criteria laid down in Regulation (EC) No 1272/2008 (i.e. CLP).