Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
Zahniser et al., 1978. Effects of N-methylaminoethanol, and N,N-dimethylaminoethanol in the diet of pregnant rats on neonatal rat brain cholinergic and phospholipid profile. Journal of neurochemistry, Vol. 30, pp. 1245-1252.
Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: combined study (repeated dose and reproduction/developmental toxicity screening)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-11 weeks
- Weight at study initiation: The weight variation of the animals used did not exceed 20 percent of the mean weight of each sex.
- Fasting period before study: no data
- Housing: individually in type M III polycarbonate cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20-24 °C,
- Humidity (%):30-70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
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
- highly deionized water
- Concentration in vehicle:0.5, 1.5 and 4.5 g/100 mL
- Amount of vehicle (if gavage): 100 mL
- Lot/batch no. (if required):
- Purity:
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
Details on analytical verification of doses or concentrations:
The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF SE. 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). The concentration control analyses revealed that the values were in the expected range of
the target concentration, i.e. were in a range of about 90.1-102.2% of the nominal concentration.
Duration of treatment / exposure:
The duration of treatment covered a 2-week pre-mating 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 (35 days for males and 55 days for females).
Frequency of treatment:
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:
0, 50, 150 and 450 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): randomized
- Other:
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
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.
- Time schedule: A cageside examination was conducted before and after treatment for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented for each animal.
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed in all animals 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 (50 x 37.5 cm with side borders of 25 cm high).
- The parameters examined are listed in the Table 1

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning).


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no

Generally, food consumption was determined 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 0
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).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OTHER:
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:
[testis weight, epididymis weight, other:]
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.
The pups were weighed one day after birth (PND 1) and on day 4 after birth.

GROSS EXAMINATION OF DEAD PUPS:
[ yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.]
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]

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.] All parental animals were sacrificed by decapitation using isoflurane anesthesia. 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. Organ weights were recorded (see Table 2).

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [3] were prepared for microscopic examination and weighed, respectively.
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:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see details on results
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
see details on results
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 (nominal)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: general, systemic toxicity
Dose descriptor:
NOAEL
Remarks:
less than
Effect level:
50 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: general, systemic toxicity
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: ferility, reproductive performance
Clinical signs:
no effects observed
Description (incidence and severity):
meets only control and low dose group
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
see details on results
Body weight and weight changes:
no effects observed
Description (incidence and severity):
meets only control and low dose group
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
meets only control and low dose group
Histopathological findings:
no effects observed
Description (incidence and severity):
meets only control and low dose group
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


OTHER FINDINGS (OFFSPRING)
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

Conclusions:
Under the conditions of the present reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance and fertility was 50 mg/kg bw/d for the parental rats. The NOAEL for general, systemic toxicity of the test substance was 50 mg/kg bw/d for females and less than 50 mg/kg bw/d for male animals based on the tubular degeneration in the kidneys of six males.
Executive summary:

Methylaminoethanol was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 50, 150 and 450 mg/kg bw/d.

The objective of the study was to detect possible effects of the test substance on the integrity and performance of the reproductive system of both sexes. Furthermore, it was intended to obtain information about the general toxicological profile including target organs and the no observed adverse effect level (NOAEL) after repeated oral administration. Control animals were dosed daily with the vehicle (highly deionized water). The duration of treatment covered a 2-week pre-mating 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.

Regarding clinical examinations, signs of general systemic toxicity were only observed at a dose level of 450 mg/kg bw/d as there were significantly lower body weights in male and female parental animals accompanied with reduced food consumption and reduced general condition in single animals in several phases of the study. Reduced food consumption and body weights during gestation in females of test group 2 (150 mg/kg bw/d) were most likely related to implantation losses.

Detailed clinical examinations in an open field, detailed observations in a functional observational battery (FOB) and measurements of motor activity did not reveal indications of test substance-induced effects in low, mid and high-dose rats. Therefore, the clonic convulsions were assessed as being incidental.

Salivation was seen after dosing in all high-dose rats. From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. Urine discoloration was also observed for all high-dose rats which was most likely related to the test compound. However, both types of findings were not considered to be adverse, toxicologically relevant effects.

Fertility was severely impaired by test-substance administration at dose levels of 150 and 450 mg/kg bw/d. Although mating (male and female mating indices) was not influenced no lifeborn pups were delivered for both test groups.

The deviated levels of clinical chemistry and haematology parameters pointed to anemia and changed liver cell metabolism. The total protein and the albumin levels were significantly higher in female rats starting at test group 1 (50 mg/kg bw/d). As these were the only deviating parameters in females of this test group the changes were regarded as treatment-related, but non-adverse. The reason for the increase of the sodium concentrations in rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) remains unclear, but a test substance-related effect could not be excluded. The higher incidences of leucocytes in the urine of rats of both sexes in test group 3 (450 mg/kg bw/d) and, additionally, in males of the test group 2 (150 mg/kg bw/d) as well as the increased incidence of higher transitional cell counts in males of test group 3 (450 mg/kg bw/d) can be regarded as signs of an affection of the urinary tract in treated rats.

Regarding pathology, after administration of the test substance the terminal body weight was significantly lower in females of test group 2 (150 mg/kg bw/d) and in males and females of test group 3 (450 mg/kg bw/d). The body weight reduction resulted in weight changes of adrenal glands, brain, heart, seminal vesicle, and thymus. Target organs were the kidney, testes, epididymides, ovaries, liver, and spleen. In kidneys and testes, tubular degeneration was dose dependent and assessed as an adverse effect. In ovaries, the occurrence of cysts and vacuolization of sex cord stroma was related to treatment and was considered to be adverse. 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 (450 mg/kg bw/d). In addition, in females of these test groups the severity of hemosiderin storage was increased. These findings are 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 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). In females, the liver enlargement correlated with a minimal central hepatocellular hypertrophy that was observed in five 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 two 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. 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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
High quality (GLP guideline study)
Additional information

Methylaminoethanol was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 50, 150 and 450 mg/kg bw/d (BASF, 2010).

The objective of the study was to detect possible effects of the test substance on the integrity and performance of the reproductive system of both sexes. Furthermore, it was intended to obtain information about the general toxicological profile including target organs and the no observed adverse effect level (NOAEL) after repeated oral administration. Control animals were dosed daily with the vehicle (highly deionized water). The duration of treatment covered a 2-week pre-mating 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.

Regarding clinical examinations, signs of general systemic toxicity were only observed at a dose level of 450 mg/kg bw/d as there were significantly lower body weights in male and female parental animals accompanied with reduced food consumption and reduced general condition in single animals in several phases of the study. Reduced food consumption and body weights during gestation in females of test group 2 (150 mg/kg bw/d) were most likely related to implantation losses.

Detailed clinical examinations in an open field, detailed observations in a functional observational battery (FOB) and measurements of motor activity did not reveal indications of test substance-induced effects in low, mid and high-dose rats. Therefore, the clonic convulsions were assessed as being incidental.

Salivation was seen after dosing in all high-dose rats. From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. Urine discoloration was also observed for all high-dose rats which was most likely related to the test compound. However, both types of findings were not considered to be adverse, toxicologically relevant effects.

Fertility was severely impaired by test-substance administration at dose levels of 150 and 450 mg/kg bw/d. Although mating (male and female mating indices) was not influenced no lifeborn pups were delivered for both test groups.

The deviated levels of clinical chemistry and haematology parameters pointed to anemia and changed liver cell metabolism. The total protein and the albumin levels were significantly higher in female rats starting at test group 1 (50 mg/kg bw/d). As these were the only deviating parameters in females of this test group the changes were regarded as treatment-related, but non-adverse. The reason for the increase of the sodium concentrations in rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) remains unclear, but a test substance-related effect could not be excluded. The higher incidences of leucocytes in the urine of rats of both sexes in test group 3 (450 mg/kg bw/d) and, additionally, in males of the test group 2 (150 mg/kg bw/d) as well as the increased incidence of higher transitional cell counts in males of test group 3 (450 mg/kg bw/d) can be regarded as signs of an affection of the urinary tract in treated rats.

Regarding pathology, after administration of the test substance the terminal body weight was significantly lower in females of test group 2 (150 mg/kg bw/d) and in males and females of test group 3 (450 mg/kg bw/d). The body weight reduction resulted in weight changes of adrenal glands, brain, heart, seminal vesicle, and thymus. Target organs were the kidney, testes, epididymides, ovaries, liver, and spleen. In kidneys and testes, tubular degeneration was dose dependent and assessed as an adverse effect. In ovaries, the occurrence of cysts and vacuolization of sex cord stroma was related to treatment and was considered to be adverse. 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 (450 mg/kg bw/d). In addition, in females of these test groups the severity of hemosiderin storage was increased. These findings are 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 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). In females, the liver enlargement correlated with a minimal central hepatocellular hypertrophy that was observed in five 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 two 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. 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.

In conclusion, under the conditions of the present reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance and fertility was 50 mg/kg bw/d for the parental rats. The NOAEL for general, systemic toxicity of the test substance was 50 mg/kg bw/d for females and less than 50 mg/kg bw/d for males based on the tubular degeneration in the kidneys of six male animals.


Short description of key information:
BASF, 2010. OECD 422 Combined Repeated-Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar rats with Methylaminoethanol (CAS No. 109-83-1; BASF, 2009).

Justification for selection of Effect on fertility via oral route:
only one reliable study available

Effects on developmental toxicity

Description of key information
Nelson et al., 1984. Comparative Inhalation Teratogenicity of Four Glycol Ether Solvents and an Amino derivative in Rats. Environmental Health perspectives, Vol., 57, pp.261-271, 1984.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
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 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 wvere 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.
Executive summary:

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 of gestation. 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. As overall result for the substance N-Methylethanolamine it can be stated that neither maternal nor fetal toxicity effects can be concluded in this study.

Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
460 mg/m³
Species:
rat
Quality of whole database:
Moderate (2 studies are available: Nelson et al., 1984 and the OECD 422 study).
Additional information

Nelson et al. (1984) performed a comprehensive investigation regarding possible maternal or fetal effects of the test substance. The chemical N-Methylethanolamine was vaporized and administered to approximately 15 pregnant rats in concentration of 150 ppm for 7 hr/day on gestation days 7 to 15, and dams were sacrificed on day 20. Fetuses were individually weighed and examined for soft-tissue anomalies and for skeletal defects.

As overall result for the substance N-Methylethanolamine it can be stated that neither maternal nor fetal toxicity effects can be concluded in this study.

The conversion of ppm in mg/m³ was performed as following: (Molecular weight of MMEA (75.11) x 150 ppm)/24.5 ; 24.5 is the volume of ideal gas at 25°C.


Justification for selection of Effect on developmental toxicity: via inhalation route:
Only one study available

Toxicity to reproduction: other studies

Additional information

Zahniser et al. investigated whether or not one could obtain a clear-cut answer to the question whether or not structural and/or metabolic alterations in the brain could be responsible for the death of the N-methylaminoethanol (MME) and N,N-dimethylaminoethanol (DME) exposed pups, observed in a previous study performed by tha same working group (Katyal and Lombardi, 1978, cited by Zahniser et al., 1978).

Pregnant rats were fed for 15 days predelivery until 15 days postpartum a choline (Ch)-deficient diet (CD diet) or a CD diet supplemented with 0.8% Ch-CI (CS), 1% MME or 1 % DME. Gestation and parturition of the pregnant rats proceeded normally. However, all the pups born of dams fed the MME diet, and most of those born of dams fed the DME diet, died within 36 h of birth. No histological or cytological alterations were detected in the brain of the pups. Levels of Ch and acetylcholine (ACh) were elevated in the brain of pups born of dams fed the MME and DME diets, but not the CS diet. It would appear, thus, that presence of MME or DME in the diet did not stimulate endogenous synthesis of Ch and ACh in adult rats. Moreover, there was no difference in the brain levels of Ch and of ACh between the CS and CD groups of dams. The content of total phospholipids in the brain of the pups was not altered by the diet fed to the dams. However, the phosphatidyl-Ch and phosphatidylaminoethanol (PAE) contents in the brain of the MME- and DMEexposed pups were markedly reduced. At the same time, significant amounts of DME, phosphatidyl-Nmonomethylaminoethanol (PMME) and of phosphatidyl-N,N-dimethylaminoethanol( PDME) were present in the same brain areas. These results are evaluated and discussed in terms of providing a cause for the death of the MME- and DME-exposed neonatal rats.

It has been concluded that, in the presence of a dietary deficiency of Ch, MME increases the demand for methyl groups and is thus more toxic than DME which is already two-thirds methylated. The observations support this conclusion in as much as supplementation of the CD diet with 1% MME, but not with 1% DME, appears to compromise the growth of the dams as well, and indicates that availability of methyl groups may be the critical factor. Alterations in brain phospholipid metabolism could also be a likely cause of death of the MME- and DME-exposed pups.

The inclusion of 1% MME or DME in the CD diet of dams limits the survival of pups after birth, most likely by interfering with the development or maturation of one or more vital systems of the fetuses.

Justification for classification or non-classification

N-methylethanolamine caused severely impaired fertility in rats treated at dose levels of 150 and 450 mg/kg bw/d in the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422; BASF, 2010). Although mating (male and female mating indices) was not influenced no lifeborn pups were delivered for both test groups. Signs of general toxicity were observed in animals of only the highest dose group (450 mg/kg bw). Changed levels of clinical chemistry parameters together with histopathological findings in organs of treated animals points to the systemic toxicity hazard by prolonged exposure. This is a summary of the most relevant findings which are very likely accountable for these effects:

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

Males (x out of 10 animals):

Target: gonades

·        Testes: tubular degeneration (8 out of 10 animals)

·        Epidymides: oligospermia (9)

 

Target: kidney

·        Kidneys: tubular degeneration (10)

·        Decreased urea clearance (mean of test group)

·        Blood in urine (mean of test group)

 

Target: liver

·        Liver: central fatty change (5)

·        Liver: peripheral fatty change (2)

·        Increased albumin level (mean of test group)

 

Target: blood

·        Spleen: extramedullar haematopoiesis (8)

·        Haemolytic anaemia (mean of test group)

 

Target: stomach

·        Forestomach: erosion/ulceration (3)

·        Glandular stomach: erosion/ulceration (2)

 

Dams (xout of 10 animals):

Target: gonades

·        Ovaries: vacuolization of sex cord stroma (10 out of 10 animals)

 

Target: kidney

·        Kidneys: tubular degeneration (9)

·        Decreased urea clearance (mean of test group)

·        Blood in urine (mean of test group)

 

Target: liver

·        Liver: central hypertrophy (9)

·        Increased albumin level (mean of test group)

 

Target: blood

·        Spleen: extramedullar haematopoiesis (8)

·        Haemolytic anaemia (mean of test group)

 

Target: stomach

·        Forestomach: erosion/ulceration (1)

 

Additional finding

·        Mesenteric lymph nodes: sinus erythrocytosis (5)

 

Test group 2 (150 mg/kg body weight/day):

Males (xanimals):

Target: kidney

·        Kidneys: tubular degeneration (10)

·        Decreased urea clearance (mean of test group)

·        Blood in urine (mean of test group)

 

Target: liver

·        Liver: peripheral fatty change (5 out of 7 animals)

 

Target: blood

·        Haemolytic anaemia (mean of test group)

Dams (xanimals):

Target: gonades

·        Ovaries: vacuolization of sex cord stroma (4 out of 7 animals)

 

Target: kidney

·        Kidneys: tubular degeneration (9)

·        Blood in urine (mean of test group)

 

Target: blood

·        Spleen: extramedullar haematopoiesis (1)

·        Haemolytic anaemia (mean of test group)

 

Additional finding

·        Mesenteric lymph nodes: sinus erythrocytosis (1 out of 2 animals)

 

Test group 1 (50 mg/kg body weight/day):

Males (xanimals):

Target: kidney

·        Kidneys: tubular degeneration (6)

 

The kidneys of 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) revealed a minimal to severe tubular degeneration (see next table) which was regarded to be treatment-related. The severity increased dose-dependently:

Tubular degeneration in the kidney:

 

Male animals

Female animals

Test group
(mg / kg bw / day)

0

 

1

(50)

2

(150)

3

(450)

0

 

1

(50)

2

(150)

3

(450)

Number of animals

10

10

10

10

10

10

10

10

Degeneration, tubular

-

6

10

10

-

-

9

9

minimal

-

3

1

1

-

-

9

7

slight

-

3

4

1

-

-

-

2

moderate

-

-

5

6

-

-

-

-

severe

-

-

-

2

-

-

-

-

 

Tubular degeneration in the testes / epididymides:

 

Maleanimals

Test group

(mg / kg bw / day)

0

1 (50)

2 (150)

3 (450)

Number ofanimals

10

10

10

10

Diffuse tubular degeneration

-

 

7

10

minimal

-

-

7

1

slight

-

-

-

1

moderate

-

-

-

5

severe

-

-

-

3

 

Tubular degeneration in kidneys and testes was dose dependent and assessed as an adverse effect. This is probably a key factor in the fertility effects. Furthermore, the effects in kidneys are probably related to anaemia. Since serum bilirubin was high, some amount of haemoglobin in the urine can be present, especially if the kidneys are damaged. It was manifested in discoloured urine of the treated animals.

Vacuolization of sex cord stroma

 

Femaleanimals

Test group

(mg / kg bw / day)

0

1 (50)

2 (150)

3 (450)

Number ofanimals

10

10

10

10

Occurrence of cysts

-

-

-

10

Vacuolization of sex cord stroma

-

-

4

10

 

The occurrence of cysts and vacuolization of sex cord stroma in ovaries was related to treatment and was considered to be adverse. This is another key factor to fertility effects.

Conclusion on classification and labelling:

Based on the findings observed in the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422; BASF, 2010) classification as toxic for reproduction is not warranted for N-methylethanolamine since the adverse effects on fertility are considered to occur secondary to the specific target organ toxicity (i.e. adverse effects on kidney and anaemia).

Classification with a“hazard category 2” for “Specific Target Organ Toxicity Repeated Exposure” (STOT RE Cat. 2) is warranted for Methyl-Monoethanolamine (MMEA; CAS 109-83-1) according to the criteria of EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulations No 1272/2008. The substance will be labelled accordingly with “H373: May cause damage to organs through prolonged or repeated exposure”. Target organs are the kidney, testes, epididymides, ovaries, liver, and spleen.

 

The rationale is as follows:

 

 1. Substance and treatment-related adverse effects on kidneys (i.e. tubular degeneration) after repeated dose exposure occur in the range of the low dose (i.e. 50 mg/kg bw/day).

 2. Substance and treatment-related adverse effects on blood (i.e. haemolytic anaemia) occurred in the range of the mid and high doses (150 and 450 mg/kg bw/day).

3. MMEA is not subject for classification as being toxic to male and female fertility. Adverse effects on fertility are considered to occur secondary to the specific target organ toxicity (i.e. adverse effects on kidney and anaemia).

 

 

Details on the rationale:

Ad 1)MMEA exerted treatment-related and adverse effects on the kidneys in males and females (i.e. tubular degeneration was observed at all doses tested. Additionally, changes in urine parameters were reported that corroborate the impaired functionality of the renal system at 150 and 450 mg/kg bw/day (e.g. occurrence of blood in the urine, reduction in urea clearance). The effects may be secondary to anaemia.

Ad 2)Haemolytic anaemia has been reported to be caused substance- and treatment-related by MMEA. A significant decrease in red blood cell count and in total haemoglobin content could be observed in the mid and high doses. In consequence, a decrease in haematocrit was observed in line with the before mentioned findings representing anaemia. Further indications for a haemolytic anaemia can be derived from the fact that an increase in hemosiderin storage in the spleens has been reported. Additionally, an increase in bilirubin levels was reported to occur in the urine indicative for the haemolytic character of the anaemia.

Ad 3)MMEA is not subject for classification for adverse effects on fertility, as the effects on testes and epididymides, as well as effects on the ovaries did not occur isolated and in the absence of other toxic effects. The adverse effects on fertility were reported to occur in the range of other significant toxic effects (i.e. kidney toxicity and haemolytic anaemia as described above). Thus, the adverse effects on reproduction affecting male and female fertility are considered to be secondary to the general toxicity effects observed and to result from a non-specific mechanism. The adverse and significant toxic effects on the kidneys occurs in the lowest dose of 50 mg/kg BW/day whereas fertility was impaired only at higher dose levels (= 150 and 450 mg/kg bw/day. Furthermore, haemolytic anaemia represents an adverse and significant effect which occurs in the same range as effects on fertility are being reported.

Reproduction and fertility have been linked with kidney failure in male rats and kidney functional impairment (Nazian and Dietz, 1987, Menjívar et al., 2000). In these available studies male fertility was reported to be impaired by liver insufficiency as a secondary consequence (e.g. chronic nephrosis, uraemia in consequence of partial nephrectomy). Especially chronic nephrosis as an umbrella term for degenerative tubular kidney disease represents a comparable situation to what has been observed for MMEA. Ortiz et al. (1999) reported decreased male fertility in consequence of chronic nephrosis.

Considering the possible mechanism, effects on the choline-homeostasis could play a role. Various alkanolamines are known to produce choline-deficiency (e.g. diethanolamine DEOA CAS 111-42-2). Choline is a vitamin-like compound with various physiological functions (i.e. building block of phospholipids and acetyl-choline, one-carbon-metabolism and DNA-methylation etc.). It could be demonstrated that certain alkanolamines exert an inhibitory effect on either choline-uptake and/or choline-metabolism. Thereby, alkanolamines cause a choline-depletion. A hallmark of choline-depletion is a fatty liver change (Zeisel, 1994). In line with this, liver enlargement concurrent with an increase in absolute absolute and relative liver weight has been reports in all dose groups after MMEA-treatment. Furthermore, minimal fatty changes and central hepatocellular hypertrophy have been observed in parallel. Similar effects have also been reported for DEOA, where choline-deficiency caused liver and kidney effects (Melnick, 1992) in repeated dose toxicity tests. In long-term studies with DEOA, liver and kidney tumours developed in mice but not in rats (NTP, 1992). In depth investigation on the possible mode-of-action revealed that the liver tumours formation could be attributed to an increase in hepatocellular proliferation probably due to a DEOA-induced choline-depletion (Lehman-McKeeman and Gamsky, 2000; Lehman-McKeeman et al., 2002). However, an increase in hepatocellular proliferation has been reported for rodent hepatocytes in vitro only whereas human primary hepatocytes did not respond. This indicates that rodent cell might be more sensitive and prone towards choline-depletion than human hepatocytes are. Thus, the human relevancy of the findings is questionable as no increase in proliferation was observed in the human hepatocytes (Stott, 2000; Kamendulis and Klaunig, 2005). Therefore, the mode-of-action of MMEA causing kidney lesions might rely on choline-depletion (as reported for various alkanoalmines as well). This mode-of-action has been demonstrated in the context of liver tumour formation to lack human relevance. It is thus concluded that MMEA-induced adverse kidney effects might arise in addition from a toxicokinetic difference with rodents being most sensitive species. This “choline” issue may also be causing some specific effect on membrane integrity that is resulting in a spectrum of toxic effects (anaemia, testes tubule degeneration, and possibly kidney tubule degeneration). This was considered to be part of the DEA toxicity spectrum as well, since some anemia was noted at the high dose levels of the DEA chronic studies.

Moreover, the dramatic reduction in male and female fertility (10 and 11%, respectively) occurred in the range of morbidity already in the high dose of 450 mg/kg bw/day. Twenty per cent of the males (2/10) were either found dead (1/10) or had to be sacrificed due a poor general status (1/10). In the mid dose (150 mg/kg bw/day) reduction in fertility was still evident in the presence of adverse and severe kidney effects and haemolytic anaemia. However, no animal died treatment related in the mid dose group.

Taken together, MMEA should be classified for specific target organ toxicity affecting kidney and blood. Effects on fertility are considered to occur as a secondary consequence and are thus not subject to classification.

 

 

References

 

Lehman-McKeeman LD, Gamsky EA (2000). Choline supplementation inhibits diethanolamine-induced

morphological transformation in Syrian hamster embryo cells: evidence for a carcinogenic mechanism. Toxicol Sci, 55, 303-10

 

Lehman-McKeeman, L. D. et al.(2002). Diethanolamine Induces Hepatic Choline Deficiency in Mice.

Toxicological sciences 67, 39-45

Kamendulis LM and Klaunig JE (2005). Species differences in the induction of hepatocellular DNA synthesis by Diethanolamnin. Toxicological Sciences 87(2),328-336.

Melnick R (1992). NTP technical report on the toxicity studies of Diethanolamine (CAS No. 111-42-2) Administered Topically and in Drinking Water to F344/N Rats and B6C3F1 Mice. Toxic Rep Ser. 20:1-D10.

Menjíar M, Cárdenas M, Ortiz G, Pedraza-Chaverrí J(2000). Fertility Diminution in Female Rats with Experimental Chronic Nephrosis. Biol reproduction 63, 1549–1554

Nazian SJ, Dietz JR (1987). Reproductive Changes during the Early Stages of Chronic Renal Insufficiency in the Mal Rat. Biol of Reproduction 37: 105-111

National Toxicology Program (1992). Toxicity Studies of Diethanolamine (CAS No. 111-42-2) Administered Topically and in Drinking Water to F344/N Rats and B6C3F1 Mice. Tech. Rep. Ser. No. 20; NIH Publication No. 92-3343), Department of Health and Human Services, Research Triangle Park, NC. Report no.: TR20.

Ortiz G, Vilchis F, Cárdenas M, Cruz C, Pedraza-Cahverris J, Menjívar M (1999). Reproduciton : Function in Male Rats with Chronic Nephrosis. Journal Reprod and Fertility 117: 223-228

Stott WT, Bartels MJ, Brzak KA, Mar M, Markham DA, Thornton CM, Zeisel SH (2000).Potential mechanisms of tumorigenic action of diethanolamine in mice. Toxicol. Lett., 114, 67-75.

Zeisel SH and Blasztajn JK (1994). Cholin and human nutrition. Ann. Rev. Nutr. 14: 269-296