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Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: combined study (repeated dose and reproduction/developmental toxicity 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: 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
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: fertility, 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.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
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, Methylaminoethanol was identified as the source chemical displaying the highest toxicity hazard when considering repeated exposure. Since no reliable conventional repeated dose oral toxicity study was available, an OECD 422 study was identified as key study and starting point for the read-across assessment.

Methylaminoethanol was given 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. The duration of treatment covered 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. At 450 mg/kg, significantly lower body weights in parental animals were accompanied with reduced food consumption and reduced general condition in single animals. Salivation was seen in all high-dose rats, likely to be related to a bad taste of the test substance or local affection of the upper digestive tract. Fertility was severely impaired at dose levels of 150 and 450 mg/kg. Although mating indices were 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 higher incidences of leucocytes in the urine in the 450 mg/kg group and in males of the 150 mg/kg group and increased incidence of higher transitional cell counts in males of 450 mg/kg group can be regarded as an affection of the urinary tract. 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 treatment-related and considered to be adverse. In the 450 mg/kg group, the infertility was linked to the reduced number of sperms (oligospermia) caused by tubular degeneration in testes. The occurrence of ovarian cysts and vacuolization of the sex cord stroma in females may also have influenced the fertility. In the 150 mg/kg group, the severity of the findings in testes or ovaries was 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 animals of middle and high dose groups. Increased hemosiderin storage in females was associated with the increased relative spleen weights in females receiving 150 mg/kg and was also present in males and females of the high dose group. They were induced in response to anaemia and related to treatment. The liver weights were dose-related increased in all animals of all treatment groups. The liver was enlarged in three males and one female receiving 150 mg/kg and in three males and five females of the 450 mg/kg group. In females, the liver enlargement correlated with a minimal central hepatocellular hypertrophy that was observed in five animals receiving 150 mg/kg and in 9 animals receiving 450 mg/kg. In males, mainly a minimal fatty change of hepatocytes was observed in two animals of the 50 mg/kg group, in 8 animals receiving 150 mg/kg, and in 7 animals of the 450 mg/kg group. The liver findings were related to treatment and considered to be adaptive. Although, there were no clear histopathological correlates for the increased liver weights, 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 were considered to be incidental or spontaneous in origin and without any relation to treatment. The NOAEL for fertility and reproductive performance was set at 50 mg/kg.

In addition, a supporting study with 2 -Aminoethanol was available. Here, a 2 -generation repeated dose dietary oral toxicity study with 2 -Aminoethanol hydrochloride (BASF SE, 2009) was used under the assumption that the observed test substance related effects were entirely attributable to 2 -Aminoethanol (and not to Hydrochloride). 2 -Aminoethanol hydrochloride was administered to groups of 25 male and 25 female healthy young Wistar rats (F0 parental generation) as a homogeneous addition to the food in different concentrations, which were adjusted regularly to obtain target dose levels of 0, 100, 300 and 1000 mg/kg bw/day. At least 75 days after the beginning of treatment, F0 animals were mated to produce a litter (F1 generation). The dose level of 1000 mg/kg bw/day caused systemic toxicity in parental females, as was indicated by reduced food consumption and/or body weight gain during gestation and lactation. In the mid and high dose F1 animals the absolute and relative kidney weights were statistical significantly increased without histopathological correlate findings. In the high dose F0 and F1 males the test substance administration led to a decrease of absolute and relative organ weights of cauda epididymidis and epididymides. Furthermore, prostate weight and the number of homogenization resistant caudal epididymidal sperm was slightly, but significantly, decreased in the F0 males. These findings were considered to be treatment-related effects, even though histomorphological correlates were missing. Based on this study with 2 -Aminoethanol hydrochloride, the NOAEL for effects on fertility was set at 300 mg/kg bw/day, which - under consideration of mass proportions (62.6 % w/w 2 -Aminoethanol in 2 -Aminoethanol hydrochloride - corresponds to a NOAEL of 188 mg/kg bw/day for 2 -Aminoethanol (300 mg/kg bw/day x 0.626).

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.

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 -Aminoethanol 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.



Short description of key information:
Based on the results of the key study with Methylethanolamine, used for a read-across assessment of the reproductive hazard of Bis[(2-hydroxyethyl)ammonium] sulfite, the NOAEL for effects on reproduction/fertility in rats was 50 mg/kg.

Justification for selection of Effect on fertility via oral route:
The key study was selected based on the source chemical displaying the highest toxicity hazard resulting from a reproduction/developmental toxicity screening test (read-across approach), i.e Methylethanolamine. An OECD 422 repeated dose dietary toxicity study with Methylethanolamine (BASF SE, 2009) was identified as key study and starting point for a read-across assessment.

Effects on developmental toxicity

Link to relevant study records

Referenceopen allclose all

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:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: sexually mature, virgin Wistar rats (Chbb :THOM (SPF)) supplied by Karl Thomae, Biberach an der Riss, Germany
- Age at study initiation: 60 days old
- Weight at study initiation: mean weight approx. 223.7 g
- Fasting period before study: none
- Housing: singly, in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm²)
- Diet: ground Kliba 343 feed rat/mouse/hamster supplied by Klingentalermuehle AG, Kaiseraugst, Switzerland, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

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

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

VEHICLE
- Concentration in vehicle: 4, 12, 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 homogeneity of the test substance was proven by visual inspection. The content of active ingredient was 99.4 % before the beginning of the study. The reanalysis of the test substance proved its stability (content: 99 .5 %).
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
day 6 - 15 of gestation
Frequency of treatment:
once daily
Duration of test:
up to day 20 p.c. (25 animals/dose group) or day 21 p.p. (15 animals/dose group)
Remarks:
Doses / Concentrations:
40, 120, 450 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
40 dams
Control animals:
yes, concurrent vehicle
Details on study design:
On day 0, the animals were assigned to the different test groups according to a randomization plan. The test substance was administered to the animals orally (by gavage) once a day during the period of major organogenesis (day 6 to day 15 p.c.) always at approx. the same time of day (in the morning). The animals of the control group were treated in the same way with the vehicle (doubly distilled water). The volume administered each day was 10 mL/kg bw. The calculation of the volume administered was based on the individual body weight determined at the beginning of the administration period (day 6 p.c.). On day 20 p.c., the first 25 animals/group were sacrificed in a randomized order and examined macroscopically. The fetuses were dissected from the uterus and further investigated with different methods. The other animals (15 dams/group) were allowed to litter and rear their pups up to day 21 p.p.. On day 21 p.p. or one of the following days the relevant dams and pups were sacrificed and examined macroscopically.
Maternal examinations:
CLINICAL OBSERVATIONS AND MORTALITY
- Time schedule: all animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited. The nesting, littering, and lactation behavior of the dams with terminal sacrifice day 21 p.p. was generally evaluated in the mornings in connection with the daily clinical inspection of the dams. Only if there were any special findings (e.g., animal could not litter, umbilical cord not cut), these specific findings were documented with the dam concerned. A check for mortality was made twice a day on working days or once a day (Saturday, Sunday or on public holidays).

BODY WEIGHT
- Time schedule: all animals were weighed on days 0, 1, 3, 6, 8, 10, 13, 15, 17 and 20 p.c.. Body weights of the animals with terminal sacrifice on day 21 p.p. were additionally determined on the day of birth and on days 4, 7, 14 and 21 p.p.. The body weight change of the animals was calculated from these results. Body weights of the animals without litter were not determined during the lactation period of the dams used in parallel. Furthermore, the corrected body weight gain was calculated for all animals with terminal sacrifice on day 20 p.c. (terminal body weight on day 20 p.c. minus weight of the uterus before it was opened minus body weight on day 6 p.c.).

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

OTHER
The littering behavior of the relevant dams was also inspected on weekdays (except holidays) in the afternoons in addition to the evaluations in the mornings. These reevaluation were documented separately, but, as before, findings were only recorded with the dams concerned. Moreover, the duration of gestation, the number of live and dead pups at birth and litter size were recorded for the animals with terminal sacrifice on day 21 p.p. For these animals the fertility and the gestation indices were calculated according to the following formulae :
fertility index = (n pregnant animals/ n mated animals) x 100
gestation index = (n animals with litters/ n pregnant animals) x 100
The values listed in the summary tables are group means determined from the fertility/gestation indices of the individual animals.
Ovaries and uterine content:
Examinations of the dams at termination
Dams with terminal sacrifice on day 20 p.c.
On day 20 p.c., the dams were sacrificed in randomized order by cervical dislocation and the fetuses dissected from the uterus. After the dams had been sacrificed, they were necropsied and assessed by gross pathology. The uterus and the ovaries were removed and the following data were recorded :

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

Calculations of conception rate and pre- and postimplantation losses were carried out:
- The conception rate (in %) was calculated according to the following formula :
conception rate = (number of pregnant animals/ number of fertilized animals) x 100

- The preimplantation loss (in % ) was calculated according to the following formula:
((number of corpora lutea - number of implantations)/number of corpora lutea) x 100

- The post implantation loss (in % ) was calculated from the following formula:
((number of implantations - number of live fetuses)/number of implantations) x 100

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

Calculations of conception rate and postimplantation loss were carried out:
- The conception rate (in %) was calculated according to the following formula :
conception rate = (number of pregnant animals/ number of fertilized animals) x 100

- The post implantation loss (in % ) was calculated from the following formula:
((number of implantations - number of live fetuses)/number of implantations) x 100
Fetal examinations:
Examination of the fetuses after dissection from the uterus
At necropsy each fetus was weighed, sexed and examined macroscopically for any external findings. The sex was determined by observing the distance between the anus and the base of the genital tubercle and was later confirmed in all fetuses fixed in Bouin's solution by internal examination. If there were discrepancies between the "external" and the "internal" sex of a fetus, the fetus was finally sexed according to the appearance of its gonads. Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes and fluids were examined. Individual placental weights were recorded. After these examinations, approximately one half of the fetuses per dam was placed in Ethyl alcohol and the other half was placed in Bouin's solution for fixation and further evaluation.

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

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

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

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

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

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

Pup number and status at delivery
All pups derived from the females were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn members of each litter. Pups which died before the first determination of their status on the day of birth were designated as stillborn pups.

Pup viability / mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by standard methods. The number and percentage of dead pups on the day of birth (day 0) and of pups dying between days 1-4, 5-7, 8-14 and 15-21 of the lactation period were determined; however, pups which died accidentally were not included in these calculations. The number of live pups/litter was calculated on the day of birth, and on lactation days 4, 7, 14 and 21. Furthermore, viability and lactation indices were calculated according to the following formulas :

Viability index (%) = (number of live pups on day 4 after birth/ number o f liveborn pups on the day of birth) x 100
Lactation index (%) = (number of live pups on day 21 after birth/ number of live pup s on day 4 after birth) x 100

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

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

Pup body weight data
The pups were weighed on the day after birth (day 1 p.p.) and on days 4, 7, 14 and 21 after birth. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). In the relevant summary tables pup body weights and pup body weight gains are listed for males, females and males + females.

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

Pup necropsy observations
After sacrifice on day 21 p.p. or one of the following days (by means of CO2 inhalation) or intercurrent death, the pups were examined externally, eviscerated and their organs were assessed macroscopically with special attention being paid to the urinary tract. After the macroscopic examination of the pups, the kidneys of each pup were placed into cassettes and fixed in 4 % formaldehyde solution for a possible further examination by light microscopy. If there were notable findings or if abnormalities were found in the daily clinical observation of the animals after their delivery, the affected animals were, if it was deemed necessary, examined additionally using appropriate methods (e.g., skeletal staining according to modified Dawson's method and/or further processing of head according to Wilson's method). The stained skeletons were evaluated under a stereomicroscope or a magnifying glass. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation (with the exception of the kidneys, see above).
Statistics:
The Dunnett-test was used for a simultaneous comparison of several dose groups with the control. The hypothesis of equal means was tested. This test was performed two-sided and was used for the statistical evaluation of food consumption, body weights and body weight change (females and pups), corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, number of corpora lutea, number of implantations, number of resorptions and number of live fetuses; proportion of preimplantation loss, postimplantation loss, resorptions and live fetuses in each litter; litter mean fetal body weight and litter mean placental weight, duration of gestation and number of pups delivered per litter. For the body weight and the body weight change of the pups the mean weight of each litter was used for the statistical analysis (statistical unit = litter).
Fisher's exact test was used for a pairwise comparison of each dose group with the control for the hypothesis of equal proportions. This test was performed one-sided and was used for statistical evaluation of the following parameters: female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings, female fertility index, gestation index, females with liveborn, stillborn and with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy.
The Wilcoxon test was used for a comparison of each dose group with the control for the hypothesis of equal medians. This test was performed one-sided and was used for the proportion of fetuses with malformations, variations, retardations and/or unclassified observations in each litter and for the proportion of affected pups per litter with necropsy observations. The significance levels were p ≤ 0.05 and p ≤ 0.01.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
The test substance caused some signs of maternal toxicity when administered by gavage to pregnant Wistar rats from days 6 - 15 of gestation at the highest dose level tested, 450 mg/kg bw/day. Maternal toxicity was substantiated by reduced food consumption, lower mean body weights and impaired body weight gain. The oral administration of the test substance at 450 mg/kg bw/day or lower dose levels had no influence on resorption rate, number of live fetuses or pups/dam, mean fetal weight or pup body weights.
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

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

Results summary

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

Test group 3 (450 mg/kg bw/day):

- statistically significantly reduced food consumption at the beginning of the treatment period (days 6 -8 p.c.), the final days of the gestation period (days 17 -20 p.c.) and during the first days of the lactation period (days 0- 4 p.p.).

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

 

Test group 2 (120 mg/kg bw/day) :

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

 

Test group 1 (40 mg/kg bw/day) :

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

 

Thus, under the conditions of this study, the test substance caused some signs of maternal toxicity when administered by gavage to pregnant Wistar rats from days 6 - 15 p.c. at the highest dose level tested (450 mg/kg bw/day). Maternal toxicity was substantiated in this dose group by a reduced food consumption, lower mean body weights and impaired body weight gain. Dose levels of 120 and 40 mg/kg bw/day did not induce any signs of maternal toxicity in the rats. There occurred no signs of developmental toxicity up to and including the highest dose level (450 mg/kg bw/day). The reproductive parameters were unaffected and neither the fetuses nor the pups showed an increased malformation rate or any indications for a substance-induced growth retardation; especially, the urinary tract of the rat fetuses/pups did not show any treatment-related findings. Based on these study results, the no observable adverse effect level (NOAEL) on the maternal organism was 120 mg/kg bw/day and 450 mg/kg bw/day for the progeny.

 

 

Detailed results

 

Maternal data:

 

Duration of pregnancy

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

Body weight

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

Food consumption

The food consumption of the high dose animals (450 mg/kg bw/day) was statistically decreased within the first days of the treatment period (days 6-8 of gestation) and also after termination of the treatment on the last days of the gestation period (17-20 of gestation). During the beginning of the lactation period (days 0-4 p.p.) there was also a slight, but statistically significant reduction in the food consumption of the high dose animals.

Clinical signs

No signs attributable to test substance administration were detected during gestation and lactation periods. During gestation, piloerection was recorded for one high dose animal on day 13. Without any dose-response relationship insufficient nesting activity was observed for several dams of all groups. During lactation, one dam of the 40 mg/kg bw/day group was found dead on day 0 p.p. after an incomplete delivery. Moreover one dam of the 120 mg/kg bw/day group had a total litter loss on day 1 after birth. All of these findings were spontaneous in nature and thus not attributed to the test substance administration.

Gross pathology

There were no substance-related observations at necropsy in any of the dams. Hydrometra (a spontaneous finding) was recorded for one female of the control group, for 2 females of the 40 mg/kg bw/day group and 3 females of the 120 mg/kg bw/day group. These animals did not become pregnant. Edema of the lungs which was related to the termination of the rats was recorded for several dams of the control, low and intermediate groups without any relation to dosing. For the one low dose female that died intercurrently during parturition, undelivered pups were found in the uterus. 

Organ weight changes, particularly effects on total uterine weight

The uterus weights, which were determined for the animals with termination on day 20 of gestation only, were not influenced by the administration of the test substance. The differences between the groups were without biological relevance and did not show any dose-response relationship.

 Fetal data:

Litter size and weights

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


Number viable (number alive and number dead)

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


Sex distribution and ratio

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

 

control: 54.6/45.4

40 mg/kg bw/day: 55.7/44.3

120 mg/kg bw/day: 46.1/53.9

450 mg/kg bw/day: 53.6/46.4

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

control: 54.4/45.6

40 mg/kg bw/day: 54.0/46.0

120 mg/kg bw/day: 45.2/54.8

450 mg/kg bw/day: 54.2/45.8

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


Organ weights

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

  

Grossly visible abnormalities, external, soft tissue and skeletal abnormalities

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


Mortality and day of death

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

 

Number pregnant per dose level

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


Number aborting

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


Number resorptions, early/late if available

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


Number of implantations

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


Pre- and post-implantation loss

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


Number of corpora lutea

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

Postnatal growth

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


Postnatal survival

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


Pup clinical observations

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

Pup necropsy observations

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

The only skeletal malformations which occurred were related to the thoracic part of the vertebral column (thoracic vertebral body/bodies dumbbell-shaped (asymmetrical) or bipartite (asymmetrical)). One or both of these malformations were found in a few fetuses of each test group including the controls without any biological relevant differences. The variations elicited were related to the ribs (shortened 13th rib(s), accessory 14th rib(s), rudimentary cervical rib(s), and the sternum (sternebra(e) of irregular shape or bipartite). These variations had no clear dose-response relationship, were found in a similar frequency in historical control data, and/or the differences between groups were without biological significance. In all groups signs of retardations (incomplete or missing ossification of vertebral bodies/arches and the sternebra(e)) were found.
 The differences between the groups, however, were not associated with the test substance administration. All of the skeletal retardations were found at a comparable frequency in the historical control data and most often a clear dose-response relationship was not present. The only statistically significant difference, an increased rate of total variations in the 120 mg/kg bw/day group, was without biological relevance because it showed no dose-dependence.
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.
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to a guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Pregnant Sprague-Dawley rats were exposed dermally to 10, 25, 75 or 225 mg/kg bw/day of 2-Aminoethanol for approximately 6 hours/day on days 6 through 15 of gestation.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc., Kingston, NY, U.S.A.
- Weight at study initiation: 250-300 g
- Housing: in wire-bottom cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C
- Humidity: 40-60 %
Route of administration:
dermal
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: shaved skin of the back
- Type of wrap: absorbant gauze pad followed by nonabsorbant cotton; an elastic bandage was wrapped securely around the animal to hold the patch in place and to prevent accidental ingestion of the test material via grooming during the exposure.

REMOVAL OF TEST SUBSTANCE
- Washing: water-dampened towel was used to wipe remaining test material off
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount applied: 1 mL/kg bw
- Constant volume or concentration used: no
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: over night
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
days 6-15 of gestation
Frequency of treatment:
6 hours/day, daily
Duration of test:
up to day 21 of gestation
Remarks:
Doses / Concentrations:
10, 25, 75, 225 mg/kg bw/day
Basis:
other: received dermal dose
No. of animals per sex per dose:
30-45 rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels selected for these studies were chosen based upon the results of dermal range-finding and teratology probe studies conducted in rats.
Maternal examinations:
CLINICAL OBSERVATIONS
- Time schedule: daily

BODY WEIGHT
- Time schedule: on gestation days 0, 3, 6-16 and 21

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

WATER CONSUMPTION: Yes

POST-MORTEM EXAMINATIONS
- Sacrifice on gestation day 21
- Organs examined: weights of liver and kidneys
Ovaries and uterine content:
The ovaries and uterine content was examined after termination.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: uteri with no visible implantations were stained with a 10 % sulfide solution.
Fetal examinations:
- External examinations: yes, all per litter
- Soft tissue examinations: yes, half per litter
- Skeletal examinations: yes, half per litter
- Head examinations: yes, half per litter

All fetuses were weighed and examined for evidence of external alterations and palate closure. At least one-half of the rat fetuses in each litter were examined for visceral alterations (Staples, 1974). The sex of all live fetuses was determined. The heads of rat fetuses not selected for skeletal examination were removed, placed in Bouin's solution, and subsequently sectioned and examined for craniofacial defects (Wilson, 1965; Van Julsingha and Bennet, 1977). All fetuses were eviscerated and stained with Alizarin red-S ( Dawson, 1926; Crary, 1962). Skeletal examinations were conducted only on the rat fetuses not selected for Bouin's examination.

References
- Crary DD (1962). Modified Benzyl alcohol clearing of Alizarin-stained specimens withut loss of flexibility. Stain Technol. 37: 124-125.
- Dawson AB (1926). A note on the staining of the skeletons of cleared specimens with Alizarin-red S. Stain Technol. 1: 123-124.
- Staples RE (1974). Detection of visceral alterations in mammalian fetuses. Teratology 9: 37 [Abstract].
- Van Julsingha EB and Bennet CG (1977). A dissecting procedure for the detection of anomalies in the foetal head. In: Methods in prenatal toxicology (Neubert D, Merker HJ and Kwasigroch TE, editors) PSG, Littleton MA, U.S.A.: 126-144.
- Wilson JG (1965). Method for administering agents and detecting malformations in experimental animals. In: Teratology: principles and techniques (Wilson JG and Warkany J, editors) Univ. of Chicago Press, Chicago, U.S.A.
Statistics:
Continuous data were evaluated for homogeneity of variance using Bartlett's test (Winer, 1971). Based upon the outcome of these tests, a parametric or nonparametric analysis of variance (ANOVA) was performed. If the ANOVA was significant, analysis by Dunnett's test (Steel and Torrie, 1960), the Wilcoxon Rank-Sum test with Bonferroni's correction (Miller, 1966), or a pooled t-test was performed as appropriate. The level of statistical significance was set a priori at a = 0.05. Nonparametric data were statistically treated using the Kruskal-Wallis test followed by the Mann-Whitney U test (Sokal and Rohlf, 1969), when appropriate. Incidence data for rats were analyzed using the Wilcoxon test as modified by Haseman and Hoel (1974). Fetal sex ratios were analyzed using a binomial distribution test (Steel and Torrie, 1960).

References
- Miller RG Jr. (1966). Simultaneous statistical inference. McGraw-Hill, New York, U.S.A..
- Sokal RR and Rohlf FJ (1969). Biometry. Freeman, San Francisco, U.S.A..
- Steel RGD and Torrie JH (1960). Principles and procedures of statistics. McGraw-Hill, New York, U.S.A..
- Winer BJ (1971). Statistical principles in experimental design. 2nd ed., McGraw-Hill, New York, U.S.A..
Details on maternal toxic effects:
Maternal toxic effects:yes

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

There were no postmortem treatment-related effects observed in any dose group. No significant differences were observed in the feed or water consumption of exposed rats relative to controls.

The body weight gain of rats given 225 mg/kg bw/day was significantly decreased during the exposure period. No effect on weight gain was observed in dams treated with lower levels. No effects on liver or kidney weights were observed at any dose level.
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
225 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Despite maternal effects observed among dams given 225 mg/kg bw/day, reproductive parameters among exposed rats were unaffected at this or lower dose levels. There were no differences in pregnancy rate, number of corpora lutea, number of implantations, resorptions, litter size, number of dead fetuses, fetal sex ratio, fetal body weight, or gravid uterine weight among any of the dose groups when compared to controls.

There were no treatment-related increases in the incidence of variations or malformations observed externally, viscerally or at skeletal examination by individual category, or in total variations or malformations when compared to controls. Among controls, the following types of malformations were observed: microphthalmia, retroesophageal right subclavian artery and an extra cervical rib. No malformed fetuses were observed in the 10 mg/kg bw/day group. Malformations observed in the 25 mg/kg bw/day group included retroesophageal right subclavian artery and an extra cervical rib. A single fetus was malformed in the 75 mg/kg bw/day dose group. This fetus had multiple craniofacial malformations consisting of micrognathia, cleft lip and soft palate, and aglossia. No malformations were observed in fetuses from dams administered 225 mg/kg bw/day.
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
450 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
460 mg/m³
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
225 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

Effects on developmental toxicity

 

No data are available for Bis[(2 -hydroxyethyl)ammonium] sulfite, for that reason assessment was performed based on read-across.

 

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 (= 460 mg/m3) 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 NOAEC was higher than 150 ppm.

 

In a prenatal developmental toxicity study performed according to OECD 414 (BASF AG, 1994), pregnant Wistar rats were exposed to the source chemical 2 -Aminoethanol by gavage at dose levels of 40, 120 or 450 mg/kg bw/day on days 6 - 15 of gestation. Signs of maternal toxicity were observed at the highest dose, manifested as reduced food consumption, lower mean body weights and impaired body weight gain. No reproductive and developmental toxicity parameters were affected. The NOAEL for developmental effects was thus at 450 mg/kg bw/day. The NOAEL for maternal toxicity by the oral route was 120 mg/kg bw/day.

In another supporting study performed comparable to an OECD 414 prenatal developmental toxicity study (Liberacki et al., 1996), pregnant Sprague-Dawley rats were exposed dermally to 10, 25, 75 or 225 mg/kg of 2-Aminoethanol for approximately 6 hours/day on days 6 through 15 of gestation. There were no treatment-related increases in the incidence of variations or malformations observed externally, viscerally or at skeletal examination by individual category, or in total variations or malformations when compared to controls. The NOAEL for maternal toxicity based on local effects including erythema and necrosis was set at 75 mg/kg and the NOAEL for developmental toxicity was higher than 225 mg/kg.

In the rabbit study performed equivalent to OECD 414 (Liberacki et al., 1996; Neeper-Bradley and Kubena, 1993), exposure was via the dermal route to 10, 25 or 75 mg/kg bw/day of 2 -Aminoethanol. The rabbits in the mid and high dose group exhibited signs of skin effects indicated by e.g. erythema, edema, necrosis, and crusting, severe at the highest dose level. No treatment-related effects were observed on reproductive and developmental toxicity parameters. The NOAEL for maternal toxicity was set at 10 mg/kg bw/day and the NOAEL for developmental toxicity was set at the highest dose level of 75 mg/kg bw/day.

Supporting studies were included for assessment which were conducted in pregnant rats with oral administration of Dipotassium disulfite (Ema et al., 1985) and 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 NOAELs for developmental toxicity were expected above the highest dose levels investigated.

Justification for classification or non-classification

Bis[(2 -hydroxyethyl)ammonium] sulfite is considered to be no subject to classification for reproduction toxicity. There are hints that a human relevance regarding the effetcs induced by amines is missing based on cholin-metabolism which is currently under evaluation.

Additional information