2-(methylamino)ethanol, compound with sulphur dioxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

toxicity to reproduction

Remarks:

other: combined repeated dose and reproduction / developmental screening

Type of information:

migrated 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11-13 weeks old
- Housing: individually, in type M III polycarbonate cages
- Diet: ground Kliba maintenance diet mouse/rat (GLP), meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-70 %
- Air changes: 10 air changes per hour
- Photoperiod: 12 hours dark / 12 hours light

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

highly deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test substance was applied as a solution. To prepare the solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then the vehicle (highly deionized water) was filled up to the desired volume, subsequently mixed using a magnetic stirrer. The test substance solutions were prepared in such intervals that the stability was guaranteed.

VEHICLE
- Vehicle: highly deionized water
- Concentration in vehicle: 5, 15, 45 mg/mL
- Amount of vehicle: 10 mL/kg bw

Details on mating procedure:

- 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

- Further matings after two unsuccessful attempts: [no]
- After successful mating each pregnant female was caged ( Pregnant females were provided with nesting material (cellulose wadding) toward the end of pregnancy.):
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in highly deionized water at room temperature for a period of 10 days was proven before the start of the administration period (Project No.: 01Y0540/078008). Homogeneity was given because the test substance was completely miscible with water and solutions were considered to be homogenous without further analysis. Concentration control analyses of the test substance preparations were performed in samples of all concentrations at the start and at the end of the administration period. The concentrations ranged from 90.1 to 102.2 % of the nominal concentrations.

Duration of treatment / exposure:

The duration of treatment covered a 2-week premating and mating period 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:

once daily (at the same time in the morning)

Details on study schedule:

- Age at mating of the mated animals in the study: [13-14] weeks

Remarks:

Doses / Concentrations:
50, 150, 450 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

10 rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose levels were selected by the Sponsor
- Rationale for animal assignment (if not random): randomized

- Other
After the acclimatization period, at least 13 days after the beignning of treatment, males and females from the same test group were mated overnight in a ratio of 1:1 or 1:2.
On study day 32, a functional observation battery and motor activity measurement were carried out in the first 5 male animals per group.
The females were allowed to litter and rear their pups until day 4 after parturition. On postnatal day 4, all pups were sacrificed and examined.
On study day 53, a functional observation battery and motor activity measurement were carried out in the first 5 female animals (with litter) per group.
From the first 5 male animals and the first 5 female animals (with litter) urinalyses were carried out on study days 34 (males) and 50 (females). Hematological and clinico-chemical examinations were carried out on study days 35 (males) and 55 (females).
At the end of the study (study day 35 for males, study day 55 for females), the animals were sacrificed after a fasting period (withdrawal of food) for at least 16-20 hours.

Positive control:

no

Parental animals: Observations and examinations:

MORTALITY
- Time schedule: a check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

CLINICAL OBSERVATIONS
- Time schedule: a cageside examination was conducted before and after treatment for any signs of morbidity, pertinent behavioural changes and signs of overt toxicity. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena.

BODY WEIGHT
- Time schedule: once a week at the same time of the day (in the morning). The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
- 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.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
- Females without a litter were weighed weekly. These body weight data were solely used for the calculations of the dose volume.
- Females after weaning (PND 4) until sacrifice were weighed once a week (for the calculation of the administration volume only)

FOOD CONSUMPTION
- Time schedule: once a week (in a period of 7 days) 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 GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined on PND 1 and 4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

HEMATOLOGY
Parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany). Furthermore differential blood smears were prepared and stained according to WRIGHT without being evaluated. The clotting analyses were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: yes (Isoflurane)
- Animals fasted: no data
- How many animals: 5 rats/sex and group
- Parameters examined: leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes, prothrombin time.

CLINICAL CHEMISTRY
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinico chemical parameters.
- Time schedule for collection of blood: in the morning
- Animals fasted: no data
- How many animals: 5 rats/sex and group
- Parameters examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), alkaline Phosphatase (ALP), gamma-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), inorganic Phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), total Bilirubin (TBIL), total protein (TPROT), Albumin (ALB), globulins (GLOB), triglycerides (TRIG), Cholesterol (CHOL), Magnesium (MG)

URINALYSIS
With the exception of volume, color, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semiquantitatively using test strips (Combur-9-test M, Roche, Mannheim, Germany) and a reflection photometer (Miditron M; Roche, Mannheim, Germany).
- Time schedule for collection of urine: overnight
- Metabolism cages used for collection of urine: yes
- Animals fasted: yes
- Parameters examined: pH, protein, Glucose, ketones, Urobilinogen, Bilirubin, blood, specific gravity, sediment, color, turbidity, volume

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: a functional observation battery was performed at the end of the administration period starting at about 10:00 h.
- Dose groups that were examined: the first 5 animals/sex and group
- Battery of functions tested: the FOB consisted of home cage observations, open field observations and sensorimotor tests/assessment of reflexes. Motor activity was assessed on the same day as the FOB.

ORGAN WEIGHTS
The following weights were determined in all parental animals sacrificed on schedule: liver, kidneys, adrenal glands, testes, epididymides, seminal vesicle, prostate, ovaries, uterus, thymus, spleen, brain, heart

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [no data]
- If yes, maximum of [all] pups/litter ; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other:] viability index was calculated as follows: (number of live pups on PND4/number of liveborn pups on the day of birth)x100. The same for sex ratio: (number of live male or female pups on day 0/ 4/number of live male and female pups on day 0/ 4)x100
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [after approximately 1 week post-mating period]
- Maternal animals: All surviving animals [after PND 4]

NECROPSY
All parental animals were sacrificed by decapitation using Isoflurane anesthesia (males: study day 35, females: study day 55). The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs. The animals, which died intercurrently or were sacrificed in a moribund state, were necropsied as soon as possible after their death and assessed by gross pathology.

HISTOLOGICAL ASSESSMENT
After the organs were fixed, histotechnical processing and examination by light microscopy was performed on following organs: trachea, lungs, liver, kidneys, spleen, adrenal glands, heart, all gross lesions, brain, spinal cord (cervical, thoracic, lumbar), sciatic nerve, thyroid glands/parathyroid glands, testes, epididymides, ovaries, uterus, vagina, prostate gland, seminal vesicles, coagulation glands, thymus, lymph nodes (axillary), lymph nodes (mesenteric), stomach (forestomach and glandular stomach), duodenum, jejunum (with Peyer’s patches), ileum, cecum, colon, rectum, urinary bladder, bone marrow (femur)

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and were sacrificed at [4] days of age. All surviving pups (after sacrifice on PND 4 by means of CO2), all stillborn pups and those pups that died before schedule, were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic
evaluation.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: all gross lesions, lungs and spinal cord (cervical, thoracic and lumbar cord) were preserved in neutrally buffered 4 % formaldehyde solution and then analyzed.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Statistics:

Food consumption, 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 pups delivered per litter, implantation sites, post implantation loss: DUNNETT-test (two-sided)
Reproduction indices and urinalysis, except color, turbidity, volume and specific gravity, 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 : FISHER'S EXACT test
Proportions of affected pups per litter with necropsy observations: WILCOXON-test (one-sided)
Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity, clinical pathology parameters, urine volume,urine specific gravity and organ weights : KRUSKAL-WALLIS test (two-sided).

Reproductive indices:

Male mating index %: (number of males with confirmed mating* /number of males placed with females)x100; *- defined by a female with vaginal sperm or with implants in utero;
Male fertility index (%): (number of males proving their fertility */number of males placed with females)x100; * - defined by a female with implants in utero;
Female mating index (%): (number of females mated */ number of females placed with males)x100; * - defined as the number of females with vaginal sperm or with implants in utero;
Female fertility index (%): (number of females pregnant */number of females mated **)x100; * defined as the number of females with implants in utero; ** defined as the number of females with vaginal sperm or with implants in utero.
Gestation index (%): (number of females with live pups on the day of birth/number of females pregnant *); * - defined as the number of females with implants in utero;
Live birth index(%): (number of liveborn pups at birth/total number of pups born)x100;
Post implantation loss (%): (number of implantations number of pups delivered/number of implantations)x100

Offspring viability indices:

Viability index (%): (number of live pups on PND4/number of liveborn pups on the day of birth)x100. The same for sex ratio: (number of live male or female pups on day 0/ 4/number of live male and female pups on day 0/ 4)x100

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

In test group 3 (450 mg/kg bw/d) one male animal (animal no. 37) was found dead within the first week of the study. One male animal (animal no. 32) of test group 3 (450 mg/kg bw/d) was sacrificed in a moribund state in study week 2. In addition, one female animal (animal no. 126) of test group 2 (150 mg/kg bw/d) was sacrificed on GD 23 because of an inability to deliver.

In test group 3 (450 mg/kg bw/d), salivation after treatment was observed in study week 1 in one male animal (animal no. 36) and in study weeks 1, 6 and 7 in six female animals. Poor general state was observed in test group 3 (450 mg/kg bw/d) in study weeks 1 and 2 in two male animals (animal nos. 32 and 36) and in study weeks 1, 6 and 7 in two female animals (animal nos. 132 and 135). In test group 3 (450 mg/kg bw/d), apathy was observed in study week 2 in one male animal (animal no. 32). Clonic convulsion was observed in test group 3 (450 mg/kg bw/d) in study week 1 in one
male animal (animal no. 39).

The detailed clinical observations on study days 0, 7, 13, 21, 28 in males and females and
additionally day 35, 42 and 49 in female animals did not reveal any additional abnormalities
in animals of test groups 0-3 (0, 50, 150 and 450 mg/kg bw/d).


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

In test group 3 (450 mg/kg bw/d) male animals’ body weight was significantly lower in week 4
and body weight change was already significantly lower between weeks 1-2 and in summary
between weeks 0-4. In test group 2 (150 mg/kg bw/d) male animals’ body weight change was
significantly lower between weeks 3-4
Body weights and body weight changes of all female animals treated with 50, 150 or 450
mg/kg bw/d were not significantly changed during premating.
During gestation body weights of female animals of test group 2 (150 mg/kg bw/d) were
significantly lower on GD 14 and 20 and of test group 3 (450 mg/kg bw/d) body weight was
even decreased on GD 20.
Body weight changes of female animals during gestation were significantly lower between
GD 0-7 in test group 1 (50 mg/kg bw/d) as well as between GD 0-7 and GD 7-14 in test group
2 (150 mg/kg bw/d). A body weight loss could be detected between GD 14-20 in test groups
2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). Consequently, the overall body weight change
between GD 0-20 was also significantly lower for these test groups.
Body weights and body weight changes of female animals treated with 50 mg/kg bw/d were
not significantly changed during lactation. During lactation, a comparison of body weight data
of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) to the control were not meaningful
as only one litter consisting of one stillborn pup existed in test group 2 (150 mg/kg bw/d) and
no pups were alive in test group 3 (450 mg/kg bw/d).
During the post-weaning period female body weights were significantly lower in test groups 2
(150 mg/kg bw/d) and 3 (450 mg/kg bw/d) in study week 6 and 7. The same was true for
females of test group 1 (50 mg/kg bw/d) in study week 7. As the terminal mean body weight
in this test group was unaffected (see section 4.4.1.1. Absolute organ weights) this change
was assessed as incidental and not related to treatment.

Significantly decreased food consumption of the male animals of test group 3 (450 mg/kg
bw/d) was observed during the first two study weeks.
Food consumption of the female rats of test group 3 (450 mg/kg bw/d) was significantly
decreased during the first study week.
During gestation the food consumption in test group 2 (150 mg/kg bw/d) was significantly
decreased between GD 14 and 20.
During lactation food consumption in test group 2 (150 mg/kg bw/d) was significantly lower
compared to the control.


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS) not applicable

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS) not examined

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS) not examined

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

The male mating index was 100% in all test groups. Fertility was proven for most of the F0 parental males of test groups 0 (control) and 1 (50
mg/kg bw/d) within the scheduled mating interval for the F1 litter. One control male and one male of test group 1 did not generate F1 pups. Furthermore, six males of test group 2 and nine males of test group 3 did not generate F1 pups. Thus, the male fertility index ranged between 11% and 90% (see Tab.4 ). For test groups 0 (control) and 1 (50 mg/kg bw/d) these findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data (see PART III, Supplement). With regard to pathological findings in epididymidis and testis (see section 4.4. Pathology) the test substance did adversely affect reproduction of the F0 males in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d).
The female mating index calculated after the mating period for F1 litter was 100% for all test groups. The mean duration until sperm was detected (GD 0) amounted to 2.4, 1.4, 2.5, and 2.9 days (0, 50, 150 and 450 mg/kg bw/d, respectively). Consequently, the differences between the test groups were assessed as being spontaneous in nature and without biological relevance. All sperm-positive rats of test groups 0 (control) and 1 (50 mg/kg bw/d) delivered pups or had implantations in utero with the following exceptions: one female (test group 0) and one female (50 mg/kg bw/d) did not become pregnant. 6 females of test group 2 (150 mg/kg bw/d), and 9 females of test group 3 (450 mg/kgbw/d) did not become pregnant. The fertility index varied between 10% and 90% (Tab. 5).
Implantation was not affected by the treatment in test group 1 (50 mg/kg bw/d) since the
mean number of implantation sites was comparable test group 0 (0 mg/kg bw/d). In test
groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) a significant reduction with only 5 and 1
implantation sites was found.The mean duration of gestation, i.e. 22.1 and 22.2 days, was similar in test groups 0 (control)
and 1 (50 mg/kg bw/d). No parturition was seen in test group 2 (150 mg/kg bw/d) except of
female No. 126 which was sacrificed on GD 23 because of an inability to deliver. Gestation
length was not calculable for test group 3 (450 mg/kg bw/d).
The gestation index varied between 89% (control group) and 100% (50 mg/kg body
weight/day). All values seen in test groups 0 (control) and 1 (50 mg/kg bw/d) reflect the normal range of
biological variation inherent in the strain of rats used for this study. All respective values were
within the range of the historical control data (PART III, Supplement) and did not show a
relation to dosing. However, a clear relation to dosing was obtained for test groups 2 (150
mg/kg bw/d) and 3 (450 mg/kg bw/d).
The mean number of F1 pups delivered per dam was not affected in test group 1 (50 mg/kg
bw/d) whereas only one pup was delivered in test group 2 (150 mg/kg bw/d) and none in test
group 3 (450 mg/kg bw/d).
The rate of liveborn pups was unaffected in test group 1 (50 mg/kg bw/d) and the live birth
index was 96%. The rate of stillborn pups was not significantly different compared to the
control group and within the range of the historical control data (PART III, Supplement).
In test group 2 (150 mg/kg bw/d) the live birth index was 0 because only one stillborn pup
was delivered.

ORGAN WEIGHTS (PARENTAL ANIMALS)

Absolute organ weights: When compared to control group 0 (set to 100%), the mean absolute weights of the organs listed in the Table 8 were significantly increased or decreased. All other mean absolute weight parameters did not show significant differences when
compared to test group 0 (control).
Relative organ weights: The terminal body weight was significantly decreased in males of test group 3 (450 mg/kg
bw/d) and in females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) resulting in
significant, secondary weight changes in various organs (Table 9)


GROSS PATHOLOGY (PARENTAL ANIMALS)

Three males of test group 3 (450 mg/kg bw/d) showed erosions or ulcers in the glandular
stomach.
The liver was enlarged in three males and one female of test group 2 (150 mg/kg bw/d) as
well as in three males and five females of test group 3 (450 mg/kg bw/d). Four males of test
group 1 (50 mg/kg bw/d) and four males of test group 2 (150 mg/kg bw/d) showed a
prominent acinar pattern of the liver.
The mesenteric lymph nodes were red discolored in one female of test group 2 (150 mg/kg
bw/d) and in two females of test group 3 (450 mg/kg bw/d).
All other gross lesions occurred either singly or were biologically equally distributed over the
control group and the treatment groups. They were considered to be incidental.

HISTOPATHOLOGY (PARENTAL ANIMALS) (see Table 8)

Kidneys: The graded severity of tubular degeneration was dose-related increased. The statistically
significant increase of the relative kidney weights in animals of test groups 2 (150 mg/kg
bw/d) and 3 (450 mg/kg bw/d) was considered to be caused by the tubular degeneration/
regeneration process.
Testes: The decrease of the absolute testes weight in males of test group 3 (450 mg/kg bw/d) was
related to the diffuse tubular degeneration.
Ovaries: In ovaries, vacuoles of different size were observed in the sex cord stroma in females of test
groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). Incidence and severity was dose-related
increased (see Table 10). In addition, one female of test group 1 (50 mg/kg bw/d), one female of test group 2 (150
mg/kg bw/d) and all females of test group 3 (450 mg/kg bw/d) showed ovarian cysts. The
occurrence of cysts in females of test group 3 (450 mg/kg bw/d) was assessed as treatmentrelated.
The cysts in each one female of test groups 1 (50 mg/kg bw/d) and 2 (150 mg/kg
bw/d) were considered to be rather incidental.
Although there was no clear histopathological correlate for the decreased absolute and
relative ovarian weights in females of test group 3 (450 mg/kg bw/d), a test substance-related
effect cannot be ruled out.
Spleen: Incidence and graded severity of extramedullary hematopoiesis were dose-related increased
in males and females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). The increased relative spleen weights in males of test group 3 (450 mg/kg bw/d) as well as in
females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) were associated with
these findings.

OTHER FINDINGS (PARENTAL ANIMALS) clinical chemistry, haematology, urinalysis, neurobehavioral observations (see under endpoint 7.5.1.)

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on reduced fertility index, reduced number of implantation sites and no delivered pups (450 mg/kg bw/day), respectively only one stillborn pup and no liveborn pups delivered (150 mg/kg bw/day).

VIABILITY (OFFSPRING)

The mean number of delivered pups per dam and the rate of liveborn and stillborn pups were
evenly distributed among test groups 0 (control) and 1 (50 mg/kg bw/d). The respective
values reflect the normal range of biological variation inherent in the strain used in this study.

The viability index as indicator for pup mortality between PND 0-4 was 100% for test groups
0 (control) and 1 (50 mg/kg bw/d). No viable pups were observed in test group 2 (150 mg/kg
bw/d) and test group 3 (450 mg/kg bw/d).

CLINICAL SIGNS (OFFSPRING)

The F1 pups did not show adverse clinical signs up to scheduled sacrifice on PND 4. In one
litter (dam No. 112 of test group 1) one pup showed a papilloma-like a skin flap. This single
observation was considered to be spontaneous in nature and not to be adverse.

BODY WEIGHT (OFFSPRING)

Mean pup body weights/pup body weight changes of all pups in test group (50 mg/kg bw/d)
were comparable to the concurrent control values. The observable differences between the
groups were assessed as being spontaneous in nature and without biological relevance.
One runt of each gender was seen in test group 0 (control) and 5 female runts were seen in
test group 1 (50 mg/kg bw/d). Both values were within the range of the biological variation
inherent in the strain of rats used for this study.

SEXUAL MATURATION (OFFSPRING) not applicable

ORGAN WEIGHTS (OFFSPRING) not examined

GROSS PATHOLOGY (OFFSPRING)

One stillborn pup of test group 1 (50 mg/kg bw/d) showed post mortem autolysis. In 3 pups of
test group 1 (50 mg/kg bw/d) and in the single stillborn pup of test group 2 (150 mg/kg bw/d)
the stomach was found empty.

HISTOPATHOLOGY (OFFSPRING) no treatment -related effects in the low dose group

Reproductive effects observed:

not specified

Table 4: Reproductive performance (male animals)

	Test group 0 (0 mg/kg bw/d)	Test group 1 (50 mg/kg bw/d)	Test group 2 (150 mg/kg bw/d)	Test group 3 (450 mg/kg bw/d)
Male fertility index [%]	90	90	40	11**

Table 5: Reproductive performance (female animals)

	Test group 0 (0 mg/kg bw/d)	Test group 1 (50 mg/kg bw/d)	Test group 2 (150 mg/kg bw/d)	Test group 3 (450 mg/kg bw/d)
Female fertility index [%]	90	90	40*	10**

* p ≤ 0.05; ** p ≤ 0.01

Table 6: Absolute organ weight (parental animals)

	Male animals			Female animals
Test group (mg/kg bw/day)	1 (50)	2 (150	3 (450)	1 (50)	2 (150	3 (450)
Terminal body weight	101%	96%	86%**	95%	93%**	85%**
Adrenal glands				96%	90%	82%**
Brain				99%	100%	96%*
Epididymides	100%	90%	68%**
Liver	113%*	121%**	129%**	105%	123%**	124%**
Ovaries				97%	99%	74%**
Testes	103%	105%	78%**
Thymus	98%	92%	67%**	88%	83%*	69%

Table 7: Relative organ weight (parental animals)

	Male animals			Female animals
Test group (mg/kg bw/day)	1 (50)	2 (150	3 (450)	1 (50)	2 (150	3 (450)
Adrenal glands	104%	102%	128%*
Brain	98%	104%	114%*	104%*	107%*	113%**
Epydidymides	94%	98%	80%**
Heart	96%	106%*	122%**	98%	105%*	116%**
Kidney	101%	110%*	126%**	108%	116%**	132%**
Liver	111%**	127%**	150%**	111%**	133%**	146%**
Ovaries				102%	106%	86%*
Seminal vesicle	104%	113%*	117%*
Spleen	102%	111%	144%**	102%	112%*	121%**
Testes	101%	110%*	91%
Thymus	97%	96%	79%*

* : p ≤ 0.05; **: p ≤ 0.01

Table 8: Histopathology (parental animals)

	Male animals			Female animals
Test group (mg/kg bw/day)	1 (50)	2 (150	3 (450)	1 (50)	2 (150		3 (450)
Kidneys	Multifocal tubular degeneration				Multifocal tubular degeneration; increase of the kidney weight
		increase of the kidney weight
Testes		diffuse tubular degeneration
Epididymides		Oligospermia
Ovaries				Ovarian cysts incidental			Ovarian cysts
Spleen		extramedullary hematopoiesis			extramedullary hematopoiesis; hemosiderin storage
Liver	Fatty change of hepatocytes				enlarged livers
Fore- and glandular stomach			Erosions or ulcers			Erosions or ulcers
Mesenteric lymph node			Sinus erythrocytosis		Sinus erythrocytosis
Thymus			reduced cellularity of cortex			reduced cellularity of cortex

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 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

Effects on fertility: via oral route (read-across)

 

In a read-across assessment, 2 -(Methylamino)ethanol was identified as the source chemical displaying the highest toxicity hazard when considering repeated exposure. In the key combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (BASF SE, 2010; 95R0540/07110), the source chemical 2 -(Methylamino)ethanol was administered to groups of 10 male and 10 female healthy young Wistar rats (F0 parental generation) by oral gavage administration at dose levels of 50, 150 or 450 mg/kg bw/day (dose volume: 10 mL/kg bw). A concurrent control group was treated with the vehicle only. The duration of treatment covered a 2 -week premating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females. At 13 days after the beginning of treatment, F0 animals were mated to produce a litter (F1 generation). Mortality, clinical signs including FOB and locomotor activity assessment, body weights, food consumption and parameters of clinical laboratory investigations (hematology, clinical chemistry, urinalysis) as well as fertility and developmental parameters were assessed/recorded during the study period. Following necropsy, the F0 generation animals were examined macroscopically and histopathologically. The weights of selected organs were determined. Regarding general repeated dose toxicity, the dose levels of 150 mg/kg bw/day and 450 mg/kg bw/day caused systemic toxicity in parental males and females, as was indicated by body weight impairment, reduced food consumption, effects on clinical pathology parameters as well as fertility (no liveborn pups), impairment of organ weights and pathological findings in the target organs (kidneys, testes, epididymides, ovaries, liver, spleen). Accordingly, the NOAEL for systemic toxicity was established at the low dose level of 50 mg/kg bw/day for female animals. For males, the low dose level of 50 mg/kg bw/day was considered to represent the LOAEL due to renal tubular degeneration noted in 6/10 males treated at this dose level.

Regarding effects on fertility, reduced fertility indices (both sexes) and a reduced number of implantation sites were noted at 150 and 450 mg/kg bw/day. At the high dose level of 450 mg/kg bw/day, no pups were delivered. At the mid dose level of 150 mg/kg bw/day, one stillborn and no liveborn pups were delivered. Accordingly, the NOAEL for effects on reproduction/fertility was set at 50 mg/kg bw/day for both sexes under the conditions of this study.

In a supporting dietary 2 -generation reproduction toxicity study (Til et al., 1972), the source chemical Disodium disulfite was shown to display a lower hazard as compared with 2 -(Methylamino)ethanol in view of systemic toxicity (including effects on fertility) following exposure by the oral route. In this study, 20 rats/sex received 0, 0.125, 0.25, 0.5, 1.0 or 2.0 % of Disodium disulfite in a Thiamine-containing diet (50 ppm) for 104 weeks (F0 and F1 generations) or for 30 weeks (F2 generation). Based on the occurrence of occult blood in faeces and changes in gastric morphology at dietary concentrations of 0.5 % or more, the NOAEL for local chronic toxicity in this study was represented by 0.25 % Disodium disulfite (or 0.215 % accounting for the loss of Metabisulfite) and was based on changes in gastric morphology (local irritation). The corrected dose level corresponded to a dose of 108 mg/kg bw/day. Because there was no evidence of systemic toxicity including effects on reproduction/fertility, the NOAEL for effects on fertility was above the highest dose of 2 % Disodium disulfite corresponding to 955 mg/kg bw/day.

In further reprotoxic studies of amines (Monoethanolamine, Diethanolamine, Triethanolamine) decreased number of implants or increased resorption rates were found. It was discussed that these effects might be mediated by a disturbed choline homeostasis 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 which might also be subject of discussion for the effects occurring after treatment with methylaminoethanol. Further studies are planned to investigate the mechanisms.

Short description of key information:
Based on the results of the key study (BASF SE, 2010; 95R0540/07110) conducted with the relevant source chemical 2-(Methylamino)ethanol, the NOAEL for both general systemic toxicity and effects on fertility was established at 50 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
The key study was selected based on the source ion(aq) displaying the highest toxicity hazard (incl. reproduction toxicity) for repeated oral exposure among the selected source chemicals (read-across approach), i.e. 2-(Methylamino)ethanol.

Effects on developmental toxicity

Description of key information

Based on the results of the key study (BASF SE, 2010; 95R0540/07110) conducted with the relevant source chemical 2-(Methylamino)ethanol, the NOAEL for developmental effects was established to be higher than 50 mg/kg bw/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

migrated 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

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11-13 weeks old
- Housing: individually, in type M III polycarbonate cages
- Diet: ground Kliba maintenance diet mouse/rat (GLP), meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-70 %
- Air changes: 10 air changes per hour
- Photoperiod: 12 hours dark / 12 hours light

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

highly deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test substance was applied as a solution. To prepare the solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then the vehicle (highly deionized water) was filled up to the desired volume, subsequently mixed using a magnetic stirrer. The test substance solutions were prepared in such intervals that the stability was guaranteed.

VEHICLE
- Vehicle: highly deionized water
- Concentration in vehicle: 5, 15, 45 mg/mL
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in highly deionized water at room temperature for a period of 10 days was proven before the start of the administration period (Project No.: 01Y0540/078008). Homogeneity was given because the test substance was completely miscible with water and solutions were considered to be homogenous without further analysis. Concentration control analyses of the test substance preparations were performed in samples of all concentrations at the start and at the end of the administration period. The concentrations ranged from 90.1 to 102.2 % of the nominal concentrations.

Details on mating procedure:

- Impregnation procedure: cohoused overnight
- M/F ratio per cage: 1:1 respectively 1:2 ratio
- Length of cohabitation: overnight, from about 16.00 h until 07.00-09.00 h of the following morning, for a maximum of 2 weeks
- Proof of pregnancy: a vaginal smear was prepared after each mating and examined for sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted "gestation day (GD) 0" and the following day "GD 1".

Duration of treatment / exposure:

The duration of treatment covered a 2-week premating and mating period 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:

once daily (at the same time in the morning)

Duration of test:

at least subchronic exposure duration for the parental generation

Remarks:

Doses / Concentrations:
50, 150, 450 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose levels were selected by the Sponsor (no justification provided in the report)

- Other
On study day 32, a functional observation battery and motor activity measurement were carried out in the first 5 male animals per group.
The females were allowed to litter and rear their pups until day 4 after parturition. On postnatal day 4, all pups were sacrificed and examined.
On study day 53, a functional observation battery and motor activity measurement were carried out in the first 5 female animals (with litter) per group.
From the first 5 male animals and the first 5 female animals (with litter) urinalyses were carried out on study days 34 (males) and 50 (females). Hematological and clinico-chemical examinations were carried out on study days 35 (males) and 55 (females).
At the end of the study (study day 35 for males, study day 55 for females), the animals were sacrificed after a fasting period (withdrawal of food) for at least 16-20 hours.

Maternal examinations:

MORTALITY
- Time schedule: a check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

CLINICAL OBSERVATIONS
- Time schedule: a cageside examination was conducted before and after treatment for any signs of morbidity, pertinent behavioural changes and signs of overt toxicity. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena.

BODY WEIGHT
- Time schedule: once a week at the same time of the day (in the morning). The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
- 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.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
- Females without a litter were weighed weekly. These body weight data were solely used for the calculations of the dose volume.
- Females after weaning (PND 4) until sacrifice were weighed once a week (for the calculation of the administration volume only)

FOOD CONSUMPTION
- Time schedule: once a week (in a period of 7 days) 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 GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined on PND 1 and 4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

HEMATOLOGY
Parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany). Furthermore differential blood smears were prepared and stained according to WRIGHT without being evaluated. The clotting analyses were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: yes (Isoflurane)
- Animals fasted: no data
- How many animals: 5 rats/sex and group
- Parameters examined: leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes, prothrombin time.

CLINICAL CHEMISTRY
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinico chemical parameters.
- Time schedule for collection of blood: in the morning
- Animals fasted: no data
- How many animals: 5 rats/sex and group
- Parameters examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), alkaline Phosphatase (ALP), gamma-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), inorganic Phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), total Bilirubin (TBIL), total protein (TPROT), Albumin (ALB), globulins (GLOB), triglycerides (TRIG), Cholesterol (CHOL), Magnesium (MG)

URINALYSIS
With the exception of volume, color, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semiquantitatively using test strips (Combur-9-test M, Roche, Mannheim, Germany) and a reflection photometer (Miditron M; Roche, Mannheim, Germany).
- Time schedule for collection of urine: overnight
- Metabolism cages used for collection of urine: yes
- Animals fasted: yes
- Parameters examined: pH, protein, Glucose, ketones, Urobilinogen, Bilirubin, blood, specific gravity, sediment, color, turbidity, volume

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: a functional observation battery was performed at the end of the administration period starting at about 10:00 h.
- Dose groups that were examined: the first 5 animals/sex and group
- Battery of functions tested: the FOB consisted of home cage observations, open field observations and sensorimotor tests/assessment of reflexes. Motor activity was assessed on the same day as the FOB.

ORGAN WEIGHTS
The following weights were determined in all parental animals sacrificed on schedule: liver, kidneys, adrenal glands, testes, epididymides, seminal vesicle, prostate, ovaries, uterus, thymus, spleen, brain, heart

NECROPSY, MACROSCOPIC EXAMINATION
All parental animals were sacrificed by decapitation using Isoflurane anesthesia (males: study day 35, females: study day 55). The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs. The animals, which died intercurrently or were sacrificed in a moribund state, were necropsied as soon as possible after their death and assessed by gross pathology.

HISTOLOGICAL ASSESSMENT
After the organs were fixed, histotechnical processing and examination by light microscopy was performed on following organs: trachea, lungs, liver, kidneys, spleen, adrenal glands, heart, all gross lesions, brain, spinal cord (cervical, thoracic, lumbar), sciatic nerve, thyroid glands/parathyroid glands, testes, epididymides, ovaries, uterus, vagina, prostate gland, seminal vesicles, coagulation glands, thymus, lymph nodes (axillary), lymph nodes (mesenteric), stomach (forestomach and glandular stomach), duodenum, jejunum (with Peyer’s patches), ileum, cecum, colon, rectum, urinary bladder, bone marrow (femur)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: yes
Examinations included:
- Gravid uterus weight: no data
- Number of corpora lutea: no
- Number of implantations: yes
- Number of early resorptions: no
- Number of late resorptions: no

Fetal examinations:

PUP NECROPSY OBSERVATIONS
All surviving pups (after sacrifice on PND 4 by means of diluted nacoren and exsanguination via vena cava), all stillborn pups and those pups that died before schedule, were examined externally, eviscerated and their organs were assessed macroscopically.

Statistics:

- Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means. Parameters analysed:
food consumption, body weight and body weight change, number of mating days, duration of gestation, number of pups per litter, implantation sites, post implantation loss

- Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions. Parameters analysed:
mating index, fertility index, 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

- Non-parametric oneway 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 WILCOXON test (two-sided) for the equal medians. Parameters analysed:
feces, rearing, grip strength, landing foot-splay, motor activity, organ weights

Indices:

Male reproduction data: mating and fertility index (%).
Female reproduction and delivery data: mating, fertility, gestation and live birth indices and post implantation loss (%)

Pups:
Viability index (%) and sex ratio

Historical control data:

Historical control data were included in the report.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
Mortality
In test group 3 (450 mg/kg bw/d) 1 male animal was found dead within the first week of the study. One male animal of test group 3 (450 mg/kg bw/d) was sacrificed in a moribund state in study week 2. In addition, 1 female animal of test group 2 (150 mg/kg bw/d) was sacrificed on GD 23 because of an inability to deliver.

Clinical signs
In test group 3 (450 mg/kg bw/d), salivation after treatment was observed in study week 1 in 1 male animal and in study weeks 1, 6 and 7 in 6 female animals. Poor general state was observed in test group 3 (450 mg/kg bw/d) in study weeks 1 and 2 in 2 male animals and in study weeks 1, 6 and 7 in 2 female animals. In test group 3 (450 mg/kg bw/d), apathy was observed in study week 2 in a single male animal. Clonic convulsion was observed in test group 3 (450 mg/kg bw/d) in study week 1 in 1 male animal. The detailed clinical observations on study days 0, 7, 13, 21 and 28 in males and females and additionally on days 35, 42 and 49 in females did not reveal any additional abnormalities in animals of all test groups.

BODY WEIGHT AND BODY WEIGHT GAIN
In test group 3 (450 mg/kg bw/d) male animals’ body weight was significantly lower in week 4 and body weight change was already significantly lower between weeks 1-2 and in summary between weeks 0-4. In test group 2 (150 mg/kg bw/d) male animals’ body weight change was significantly lower between weeks 3-4. Body weights and body weight changes of all female animals treated with 50, 150 or 450 mg/kg bw/d were not significantly changed during premating. During gestation body weights of female animals of test group 2 (150 mg/kg bw/d) were significantly lower on GD 14 and 20 and of test group 3 (450 mg/kg bw/d) body weight was even decreased on GD 20. Body weight changes of female animals during gestation were significantly lower between GD 0-7 in test group 1 (50 mg/kg bw/d) as well as between GD 0-7 and GD 7-14 in test group 2 (150 mg/kg bw/d). A body weight loss could be detected between GD 14-20 in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). Consequently, the overall body weight change between GD 0-20 was also significantly lower for these test groups. Body weights and body weight changes of female animals treated with 50 mg/kg bw/d were not significantly changed during lactation. During lactation, a comparison of body weight data of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) to the control were not meaningful as only one litter consisting of one stillborn pup existed in test group 2 (150 mg/kg bw/d) and no pups were alive in test group 3 (450 mg/kg bw/d). During the post-weaning period female body weights were significantly lower in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) in study week 6 and 7. The same was true for females of test group 1 (50 mg/kg bw/d) in study week 7. As the terminal mean body weight in this test group was unaffected this change was assessed as incidental and not related to treatment.

FOOD CONSUMPTION
Significantly decreased food consumption of the male animals of test group 3 (450 mg/kg bw/d) was observed during the first two study weeks. Food consumption of the female rats of test group 3 (450 mg/kg bw/d) was significantly decreased during the first study week. During gestation the food consumption in test group 2 (150 mg/kg bw/d) was significantly decreased between GD 14 and 20. During lactation food consumption in test group 2 (150 mg/kg bw/d) was significantly lower compared to the control.

HEMATOLOGY
At the end of the administration period red blood cell counts (RBC), hemoglobin concentrations and hematocrit values were decreased in rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). Additionally, the hematocrit values were significantly decreased in females and males of test group 1 (50 mg/kg bw/d). This decrease compared to the controls was below 10 % (males: 5 %, females: 7 %), and it was the only dose-dependently changed red blood cell parameter in this test group. Therefore, the hematocrit decrease in rats of test group 1 (50 mg/kg bw/d) was regarded as treatment-related but not adverse. The mean corpuscular volume (MCV) was decreased in male rats of all treatment groups (not significantly changed in test group 3 [450 mg/kg bw/d]). The measured MCV and RBC values were used to calculate the hematocrit values. In male rats of test group 1 (50 mg/kg bw/d) the MCV reflected the decreased hematocrit value because the RBC was not changed. Therefore, the decreased MCV in these rats was regarded as treatment-related, but not adverse as mentioned above. In female rats of test group 3 (450 mg/kg bw/d) the relative reticulocyte counts were increased. No significant change was observed in the total white blood cell counts (WBC) of treated rats. However, some changes in the relative and absolute differential blood cell counts were measured (males: increased relative neutrophil counts and decreased relative eosinophil counts in test group 3 [450 mg/kg bw/d], decreased relative monocyte counts in test group 2 [150 mg/kg bw/d]; females: decreased absolute eosinophil counts in test group 3 [450 mg/kg bw/d], decreased relative neutrophil counts and increased relative lymphocyte counts in test group 2 [150 mg/kg bw/d]). These changes were regarded as being incidental and not treatment-related because they were not dose-dependently changed and not consistent in both sexes. The prothrombin time was shortened in rats of both sexes of test group 3 (450 mg/kg bw/d)
and, additionally, in females of test group 2 (150 mg/kg bw/d).

CLINICAL CHEMISTRY
Liver enzyme activity was not changed in male and female rats of any test substance-treated group. The urea levels were increased in males of test group 2 (150 mg/kg bw/d) and in rats of both sexes in test group 3 (450 mg/kg bw/d). The total Bilirubin concentrations were significantly higher in rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). The total protein and the Albumin levels were increased in females of test group 1 (50 mg/kg bw/d) and higher (total protein level was not significantly increased in test group 3 [450 mg/kg bw/d]), although the increases were not dose-dependent. In males the total protein levels were significantly increased in test groups 1 (50 mg/kg bw/d) and 2 (150 mg/kg bw/d) and the Albumin concentrations in test group 2 (150 mg/kg bw/d), only. These parameters were not changed dose-dependently, and the deviated values were within the historical control ranges (total protein: 62.45-69.74 g/L; Albumin 36.12-39.76 g/L). Therefore, these deviations were regarded as non-adverse effects.
The Sodium concentrations were increased in rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) and, additionally, in males of test group 1 (50 mg/kg bw/d). The Sodium mean in males at least of the low dose group was within the historical control range (140.9-147.1 mmol/L). Apart from this, only this electrolyte level was deviated in test group 1 (50 mg/kg bw/d). Therefore, the Sodium levels increase at least in males of the low dose group was regarded as a non-adverse effect. In males of test groups 1 (50 mg/kg bw/d) and 2 (150 mg/kg bw/d) the Cholesterol levels were decreased. The parameter was not changed dose-dependently, and such deviation was not observed in females. Therefore, the Cholesterol levels decrease in males of test groups 1 (50 mg/kg bw/d) and 2 (150 mg/kg bw/d) was regarded as non-adverse. In treated females the Potassium concentrations were significantly higher in test group 1 (50 mg/kg bw/d), the Creatinine levels were higher in test group 2 (150 mg/kg bw/d) and the Magnesium concentrations were increased in test groups 1 (50 mg/kg bw/d) and 2 (150 mg/kg bw/d). These values were not changed dose-dependently, and the deviations of these parameters were not measured in male rats. Therefore, these changes were regarded as incidental rather than treatment related.

URINALYSIS
In rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) the incidence of blood (haemoglobin) was found higher compared to the controls (in females of test group 3 not significant). Additionally, the incidence of higher leucocyte counts in the urine sediment was significantly increased in males of test group 2 (150 mg/kg bw/d). However, no significantly higher leucocyte counts were found in the urine sediment of rats of both sexes of test group 3 (450 mg/kg bw/d). In males of test group 3 (450 mg/kg bw/d), the incidence of higher transitional cell counts was increased.

The urine was discolored almost in all the males and females of test group 3 (450 mg/kg bw/d) from study week 1 onwards.

NEUROBEHAVIOUR
No test substance-related or spontaneous findings in male and female animals of all test groups during the home cage observation were observed. The open field observations did not reveal any test substance-related findings in male and female animals of all test groups. In sensorimotor test/assessment of reflexes, there were no test substance-related findings in male and female animals of all test groups. Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore, these observations were considered as being incidental. There were no significant deviations concerning the overall motor activity (summation of all intervals) in the male and female animals of all test groups in comparison to the concurrent control group. Regarding single intervals, in males of test groups 1 and 2 (50 and 150 mg/kg bw/d) two isolated significantly increased values were measured at interval 4. These findings were considered as being incidental since the overall motor activity was not changed and no findings were observed for female animals.

ORGAN WEIGHTS
The terminal body weight was significantly decreased in males of test group 3 (450 mg/kg bw/d) and in females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) resulting in significant, secondary weight changes in various organs, i.e. in decreased absolute adrenal and brain weights in females of test group 3 (450 mg/kg bw/d), in decreased absolute and relative thymus weights in males of test group 3 (450 mg/kg bw/d), in decreased absolute thymus weights in females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d), in increased relative brain and heart weights in males and females of test group 3 (450 mg/kg bw/d), in increased relative weights of adrenal glands and seminal vesicles in males of test group 3 (450 mg/kg bw/d) as well as in increased relative brain weights in females of test group 2 (150 mg/kg bw/d). The increased relative weights of heart and seminal vesicles in males of test group 2 (150 mg/kg bw/d) and the increased relative brain weight in females of test group 1 (50 mg/kg bw/d) were related to the slightly but not significantly decreased terminal body weights in these animals (-4 % and -5 %, respectively).

The increased relative kidney weights in males and females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d), the decrease of the absolute and relative ovarian weight in females of test group 3 (450 mg/kg bw/d), the increased liver weights of males and females in all test substance-treated test groups, and the increased relative spleen weights in males of test group 3 (450 mg/kg bw/d) as well as in females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) were considered to be treatment-related.

Although the relative testes weight in males of test group 3 (450 mg/kg bw/d) was only slightly but not significantly decreased (-9 %), the reduction of the absolute testes weight was assessed as treatment-related. The increase of the relative testes weight in males of test group 2 (150 mg/kg bw/d) was related to the slightly but not significantly decreased terminal body weight in these animals.

The decreased absolute and relative weights of epididymides in males of test group 3 (450 mg/kg bw/d) were related to treatment. The decrease of the absolute weight of epididymides in males of test group 2 (150 mg/kg bw/d) was considered to be incidental, because there were no histopathological correlates and the relative weights did not show significant weight changes.

GROSS PATHOLOGY
Three males of test group 3 (450 mg/kg bw/d) showed erosions or ulcers in the glandular stomach. The liver was enlarged in 3 males and 1 female of test group 2 (150 mg/kg bw/d) as well as in 3 males and 5 females of test group 3 (450 mg/kg bw/d). Four males of test group 1 (50 mg/kg bw/d) and 4 males of test group 2 (150 mg/kg bw/d) showed a prominent acinar pattern of the liver.
The mesenteric lymph nodes were red discolored in 1 female of test group 2 (150 mg/kg bw/d) and in 2 females of test group 3 (450 mg/kg bw/d). All other gross lesions occurred either singly or were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental.

FERTILITY
At the high dose level of 450 mg/kg bw/day, the male fertility index was reduced to 11 % and the female fertility index was reducet to 10 %. Further a reduced number of implantation sites was observed, and no pups were delivered. Mating (male and female mating indices) was not influenced.
At 150 mg/kg bw/day, fertility indices were reduced to 50 % in males and females. Further a reduced number of implantation sites was observed and only one stillborn pup and no liveborn pups were delivered. Mating (male and female mating indices) was not influenced.
No impairment of fertility was observed in the 50 mg/kg bw/day dose group.

HISTOPATHOLOGY
Target organs were the kidney, testes, epididymides, ovaries, liver and spleen.
In kidneys, a multifocal degeneration was observed in proximal tubular cells at the transition of cortex to medulla in males of all treatment groups as well as in females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). The severity increased dose-dependently. The tubular degeneration resulted in increased kidney weights in males and females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). The occurrence of tubular degeneration in kidneys was related to treatment and assessed as an adverse effect. In addition, 9/10 females of test group 3 (versus two control females) showed a mostly minimal or slight multifocal mineralization in the papillae. A treatment-related effect could not be ruled out, but was considered to be non-adverse.
In the testes, a diffuse tubular degeneration was observed in 7 males (minimal) of test group 2 (150 mg/kg bw/d) and in all males (mostly moderate or severe) of test group 3 (450 mg/kg bw/d). The tubular degeneration was associated with the decreased absolute weight of testes in males of test group 3 (450 mg/kg bw/d). The occurrence of diffuse tubular degeneration in testes was considered to be treatment-related and adverse. In 9 males of test group 3 (450 mg/kg bw/d), the diffuse tubular degeneration of testes caused a mostly severe oligospermia in the epididymides. The oligospermia was linked to the decreased absolute and relative weights of epididymides in these males.
In ovaries, vacuoles of different size were observed in the sex cord stroma in females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) showing dose-response relationship in incidence and severity. In addition, all females of test group 3 (450 mg/kg bw/d) showed ovarian cysts. The occurrence of cysts and vacuolization of sex cord stroma was related to treatment and was considered to be adverse. Although there were no clear histopathological correlates for the decreased absolute and relative ovarian weights in females of test group 3 (450 mg/kg bw/d), a test substance-related effect could not be ruled out. In test group 3 (450 mg/kg bw/d), the infertility was linked to the reduced number of sperms (oligospermia) caused by tubular degeneration in testes. In addition, the occurrence of ovarian cysts and vacuolization of the sex cord stroma in females may have influenced the fertility. In test group 2 (150 mg/kg bw/d), the severity of the findings in testes or ovaries was only minimal or slight and the findings did not occur in all infertile animals. Nevertheless, these lesions may have affected fertility.
In the spleen, a dose-related increase in incidence and severity of extramedullary hematopoiesis occurred in males and females of test groups 2 (150 mg/kg bw/d) and 3 (450mg/kg bw/d). In addition, in females of these test groups the severity of hemosiderin storage was increased. These findings were associated with the increased relative spleen weights in females of test group 2 (150 mg/kg bw/d) as well as in males and females of test group 3 (450 mg/kg bw/d). They were induced in response to anaemia and related to treatment.
The liver weights were dose-related increased in males and females of all treatment groups. The liver was enlarged in 3 males and 1 female of test group 2 (150 mg/kg bw/d) as well as in 3 males and 5 females of test group 3 (450 mg/kg bw/d). In females, the liver enlargement correlated with a minimal central hepatocellular hypertrophy that was observed in 5 animals of test group 2 (150 mg/kg bw/d) and in 9 animals of test group 3 (450 mg/kg bw/d). In males, mainly a minimal fatty change of hepatocytes was observed in 2 animals of test group 1 (50 mg/kg bw/d), in 8 animals of test group 2 (150 mg/kg bw/d), and in 7 animals of test group 3 (450 mg/kg bw/d). The liver findings were related to treatment and considered to be adaptive. Although, there were no clear histopathological correlates for the increased liver weights in males of all treatment groups and in females of test group 1 (50 mg/kg bw/d), a test substance-related effect could not be ruled out.
In test group 3 (450 mg/kg bw/d), erosions or ulcers occurred in the forestomach of 3 males and of 1 female. In the glandular stomach, erosions/ulcers were observed in 3 males of test group 3 (450 mg/kg bw/d) in comparison to 1 control male. A test substance related effect could not be ruled out.
Intrasinusoidal erythrocytes (sinus erythrocytosis) occurred in the mesenteric lymph nodes of 1 female (minimal) in test group 2 (150 mg/kg bw/d) and of 1 male and 3 females (slight) in test group 3 (450 mg/kg bw/d). Intrasinusoidal erythrocytes (sinus erythrocytosis) can result from a lymph node, which drains a region of hemorrhage. This can also be an artifact that results from tissue dissection during necropsy. In this study, there was no correlation between erosion/ulcer in the stomach and erythrocytosis of the mesenteric lymph node (findings occurred in different animals). However, a treatment-related effect could not be ruled out but was assessed as non-adverse.
All further findings occurred either singly or were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
VIABILITY
The viability index as indicator for pup mortality between PND (postnatal day) 0-4 was 100 % for test groups 0 (control) and 1 (50 mg/kg bw/d). No viable pups were observed in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d).

PUP NUMBER AND STATUS AT DELIVERY
The mean number of delivered pups per dam and the rate of liveborn and stillborn pups were evenly distributed among test groups 0 (control) and 1 (50 mg/kg bw/d). The respective values reflect the normal range of biological variation inherent in the strain used in this study.

SEX RATIO
The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between test groups 0 (control) and 1 (50 mg/kg bw/d).

CLINICAL SIGNS
The F1 pups did not show adverse clinical signs up to scheduled sacrifice on PND 4. In one litter (dam No. 112 of test group 1, 50 mg/kg bw/d) one pup showed a papilloma-like skin flap. This single observation was considered to be spontaneous in nature and not to be adverse.

BODY WEIGHT
Mean pup body weights/pup body weight changes of all pups in test group 1 (50 mg/kg bw/d) were comparable to the concurrent control values. The observable differences between the groups were assessed as being spontaneous in nature and without biological relevance. One runt of each gender was seen in test group 0 (control) and 5 female runts were seen in test group 1 (50 mg/kg bw/d). Both values were within the range of the biological variation inherent in the strain of rats used for this study.

GROSS PATHOLOGY
One stillborn pup of test group 1 (50 mg/kg bw/d) showed post mortem autolysis. In 3 pups of test group 1 (50 mg/kg bw/d) and in the single stillborn pup of test group 2 (150 mg/kg bw/d) the stomach was found empty.

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Reference 2

Endpoint:

developmental toxicity

Type of information:

migrated 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:

other: no guideline study, well described publication

Principles of method if other than guideline:

The chemical N-Methylethanolamine was vaporized and administered to approximately 15 pregnant rats in one to three concentrations for 7 hr/day on gestation days 7 to 15, and dams were sacrificed on day 20. Fetuses were individually weighed, and two-thirds of them were fixed in Bouin's solution and examined for soft-tissue anomalies. The other one-third were fixed in alcohol, stained with Alizarin Red and examined for skeletal defects.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Virgin female and male Sprague-Dawley rats specified to be free of mycoplasma and Sendai virus and of internal and external parasites (Charles River Breeding Laboratories, Wilmington, MA) were acclimated to a 12-hr light-dark cycle (lights on at 6 am) and to a temperature of 24 + 2°C for 2 weeks. The humidity, not controlled, typically was in the range of 40 + 20%. Purina Lab Chow and tap water were available ad libitum except when pregnant animals were in exposure chambers. Bedding consisted of cleaned, heattreated sawdust from a local supplier (Absorb-Dri, Tasty Foods, Cincinnati, OH).
Females were placed alone in 38 x 33 x 17-cm polycarbonate cages with filter tops.

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

Pregnant females were transported from the animal quarters to the inhalation chambers in their home cages with filter tops (Hazleton Systems, Aberdeen, MD). They were placed individually in 13 x 25 x 189-cm stainless steel wire mesh cages within exposure chambers.
Air flow through the chambers provided approximately four air changes per minute.
Exposures were conducted sequentially in one or two chambers, with a third chamber for sham exposure of control subjects.
Control animals were placed in similar chambers for the same hours as the exposed animals; a pooled group of controls (N = 34) served as the comparison group for the first three chemicals examined. Another group of 15 controls served as the comparison group for the last two chemicals examined, as these groups were exposed at a later time (approximately 6 months later) than the first three.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations within the exposure chambers as measured by the infrared analyzer were relatively close to those obtained from gas chromatography (Table 2).

Details on mating procedure:

Males weighing over 300 g were placed individually into a cage with three females weighing 200 to 300 g. Vaginal smears were taken each morning, and the presence of sperm marked day zero of gestation.

Duration of treatment / exposure:

7 hours (animals were left in the chamber for at least one additional hour blow-off time after vapor generation terminated)

Frequency of treatment:

Exposures, as outlined above, were conducted 7 hr/day, and the animals were left in the chamber for at least one additional hour blow-off time after vapor generation terminated. They were then returned in their individual housing cages to the animal quarters, where water bottles were replaced. Exposures were conducted on gestation days 7-15.

Duration of test:

Exposures were conducted on gestation days 7-15.
15 days of gestation.
On day 20 of gestation, dams were sacrificed.

Remarks:

Doses / Concentrations:
150.0 +/- 15.2 ppm
Basis:
other: vapor generated, by gas chromatography

No. of animals per sex per dose:

approximately 15 pregnant rats

Control animals:

yes

Details on study design:

Controls: three solvents were compared with a pooled group (N = 34) of sham-exposed controls, and the remaining two were compared with a group of 15 controls.

Maternal examinations:

Feed and water intake and maternal weight were recorded weekly (i.e., on days 7, 14, and 21); any other signs of maternal toxicity were noted daily.
On day 20 of gestation, the females were individually weighed and euthanized by chloroform asphyxiation.

Ovaries and uterine content:

The entire uterus was removed and numbers of resorption sites (classified as early, middle or late) and live fetuses were determined.

Fetal examinations:

Fetuses were serially removed, blotted of excess fluids, weighed, examined for external malformations and external sex determined.
One third of the fetuses were randomly selected and placed in 95% ethanol, and the remainingfetuses were placed in Bouin's solution. After being in
the Bouin's solution for at least 1 week, these fetuseswere examined for visceral abnormalities using Wilson's razor blade sectioning technique. The viscera wereexamined with the aid of a dissecting microscope. A representative sample of sections with malformations was identified by dam number and saved in 70% alcohol.

Fetuses were examined for skeletal defects by using a modified Staples technique. They were fixed in 95% alcohol, eviscerated and macerated in 2% KOH/Alizarin Red S solution. The fetuses were further macerated and cleared in the appropriate solutions of 2% KOH/glycerin (60:40, 40:60, 20:80) and stored in 100% glycerin. A crystal of thymol was added to each storage vial to retard fungal growth. Storage vials were individually identified by dam number.

Statistics:

Numbers of implants and proportions of resorptions were independently analyzed by using a Kruskal-Wallis test corrected for ties, with subsequent multiple comparisons to determine where the differences occurred. Analysis of pup weights involved a mixed model analysis of covariance (with the number of live pups in the litter as the covariate) using maximum likelihood estimation. The model was mixed, since there was both within-litter and between-litter variation. Subsequently, pairwise comparisons between the pooled control group and each treatment group were performed. Incidence oftotal defects and of total variants were compared using a Kruskal-Wallis test with multiple comparisons with the litter as the experimental unit and the level of significance at p< 0.05.

Details on maternal toxic effects:

Maternal toxic effects:not examined

Details on maternal toxic effects:
At 150 ppm N-Methylethanolamine (mean concentration from 28 silica gel tubes, one per day, analyzed in duplicate = 150.0 ppm), no maternal or
fetal toxicity was observed.

Dose descriptor:

NOAEC

Effect level:

150 ppm (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
At 150 ppm N-Methylethanolamine (mean concentration from 28 silica gel tubes, one per day, analyzed in duplicate = 150.0 ppm), no maternal or
fetal toxicity was observed.

Dose descriptor:

NOAEC

Effect level:

150 ppm (nominal)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Low vapor pressure also prevented our generating high concentrations of 2-MAE. At 150 ppm 2-MAE (mean concentration from 28 silica gel tubes, one per day, analyzed in duplicate = 150.0 ppm), no maternal or fetal toxicity was observed (Tables 8-10).

Finally, the lack of teratogenic response of 2-methylaminoethanol was interesting and from a mechanistic or theoretical point of view, would merit follow up using a different route of exposure. At first glance, one might expect that its biotransformation would be similar to that of 2-ME. However, our results of no maternal or fetal toxicity at 150 ppm 2-MAE suggest that this may not be the case; since the amine is likely more lipidsoluble and less water-soluble than the methoxy portion, the absorption and excretion of the 2-MAE is likely quite different from that of 2-ME. Thus it would be of interest to see if a higher dose of 2-MAE would be teratogenic, though a route other than inhalation would be required, since the vapor concentration we used was near the saturation point.' This lack of teratogenicity at three times the concentration of a teratogenic level of its structurally similar glycol ether, points to a relatively strict structural requirement to produce teratogenic effects.

We observed that embryotoxicity decreases as alkyl chain length increases, similar to observations with testicular atrophy.

Conclusions:

In this study N-Methylethanolamine showed neither maternal nor fetal toxicity effects.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

460 mg/m³

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Effects on developmental toxicity: via oral route (read-across)

In a read-across assessment, 2 -(Methylamino)ethanol was identified as the source chemical displaying the highest toxicity hazard when considering repeated exposure. In the key combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (BASF SE, 2010; 95R0540/07110), the source chemical 2 -(Methylamino)ethanol was administered to groups of 10 male and 10 female healthy young Wistar rats (F0 parental generation) by oral gavage administration at dose levels of 50, 150 or 450 mg/kg bw/day (dose volume: 10 mL/kg bw). A concurrent control group was treated with the vehicle only. The duration of treatment covered a 2 -week premating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females. At 13 days after the beginning of treatment, F0 animals were mated to produce a litter (F1 generation). Mortality, clinical signs including FOB and locomotor activity assessment, body weights, food consumption and parameters of clinical laboratory investigations (hematology, clinical chemistry, urinalysis) as well as fertility and developmental parameters were assessed/recorded during the study period. Following necropsy, the F0 generation animals were examined macroscopically and histopathologically. The weights of selected organs were determined. Regarding general repeated dose toxicity, the dose levels of 150 mg/kg bw/day and 450 mg/kg bw/day caused systemic toxicity in parental males and females, as was indicated by body weight impairment, reduced food consumption, effects on clinical pathology parameters as well as fertility (no liveborn pups), impairment of organ weights and pathological findings in the target organs (kidneys, testes, epididymides, ovaries, liver, spleen). Accordingly, the NOAEL for systemic toxicity was established at the low dose level of 50 mg/kg bw/day for female animals. For males, the low dose level of 50 mg/kg bw/day was considered to represent the LOAEL due to renal tubular degeneration noted in 6/10 males treated at this dose level.

Regarding effects on fertility, reduced fertility indices (both sexes) and a reduced number of implantation sites were noted at 150 and 450 mg/kg bw/day. At the high dose level of 450 mg/kg bw/day, no pups were delivered. At the mid dose level of 150 mg/kg bw/day, one stillborn and no liveborn pups were delivered. Accordingly, the NOAEL for effects on reproduction/fertility was set at 50 mg/kg bw/day for both sexes under the conditions of this study. On the same basis, the NOAEL for developmental toxicity (teratogenicity) was set at 50 mg/kg bw/day, i.e. the highest dose level at which no maternal toxicity was noted. Of note, there were no indications for a teratogenic potential of the source chemical 2 -(Methylamino)ethanol.

In a supporting study conducted in pregnant rabbits with oral administration of Disodium disulfite (Anonymous, 1974) during the phase of organogenesis the number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the numbers occurring spontaneously in the concurrent controls. Thus, the NOAEL for developmental toxicity was expected above the highest dose level investigated.

Effects on developmental toxicity: via inhalation route (read-across)

In a supporting study (Nelson et al., 1984), the source chemical 2 -(Methylamino)ethanol was vaporized and administered to approximately 15 pregnant rats for 7 hours/day on gestation days 7 -15 at 150 ppm (=460 mg/m3) by whole body inhalation exposure. The dams were sacrificed on day 20 of gestation. Fetuses were individually weighed, and two-thirds of them were examined for soft-tissue anomalies. The other one-thid was examined for skeletal defects. Under the conditions of this study, neither maternal nor fetal toxicity was observed at the single concentration tested.

Justification for selection of Effect on developmental toxicity: via oral route:
The key study was selected based on the source ion(aq) displaying the highest toxicity hazard (incl. developmental toxicity) for repeated oral exposure among the selected source chemicals (read-across approach), i.e. 2-(Methylamino)ethanol.

Justification for classification or non-classification

2-(Methylamino)ethanol, compound with Sulfur dioxide is considered to be no subject to classification for reproduction toxicity. There are hints that a human relevance regarding the effetcs induces by amines is missing based on cholin-metabolism which is currently under evaluation.