Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Fertility studies and developmental toxicity studies were conducted with the read-across substance as well as with the dissociation products of MEA-LAS. Overall, results from these studies indicate no adverse effect on the fertility of the parenteral animals and development of the offsprings.

Link to relevant study records

Referenceopen allclose all

Endpoint:
three-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of this study was to evaluate linear alkylbenzene sulphonate sodium salt (LAS) having C10 - 14 chain length distribution for reproductive toxicity potential in rats. Rats were divided into 4 groups and administered 0 (control), 0.02, 0.1 and 0.5% LAS added to their diets. Each group consisted of 50 males and 50 females randomized according to their litter and weight. After administration of LAS for 84 days when the parent animals (Po) were 107 – 112 days old, 20 female rats from each group were mated with 20 male rats from the same group. All females were separated from males on completion of seventeen days and placed in the opaque plastic litter boxes containing clean, dry saw dust and sufficient paper for nesting. Litters were examined for deformities and number of pups were counted. The first litters of F1a and F2a generation were sacrificed at 21 days of age and 10 days after sacrifice of litters, all females were remated with different males from same group to obtain the F1b generation. Twenty males and females of each group were selected at weaning to continue their respective diets and to be used for further reproduction studies and remaining weanling rats were examined and sacrificed. Reproduction studies on the F1b and F2b generations were started when the rats were 80 to 85 days old and were continued until the F3b generation was weaned. Average body weight, feed consumption and feed efficiency were recorded on weekly basis for the first 8 weeks for F1a and F2b generations. Once the reproductive studies were completed, five male and five female rats from each parenteral groups (F1a and F2b) were selected for necropsy and body weight, organ to body weight ratios were recorded. Routine hematology and histology studies were also performed. Weanling animals from F3a generation were treated similarly.
GLP compliance:
no
Remarks:
(predates GLP)
Limit test:
no
Species:
rat
Strain:
other: Charles River
Sex:
male/female
Details on test animals and environmental conditions:
Details on test animals and environmental conditions
TEST ANIMALS
- Age at study initiation:
a) Parent animals (Po): 107 – 112 days
b) F1b and F2b generation: 80 – 85 days
- Parent animals body weight at study initiation: 59.4 - 59.9 g (males) and 57.0 - 57.3 g (females)
- Housing:
a) Females after mating were placed in opaque plastic boxes containing clean, dry sawdust and paper for nesting
b) Weanling rats: Individual wire bottom cages
- Diet: Purina rat chow; ad libitum
- Drinking water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 76 ± 3 °F
- Humidity: 50 ± 5 %
- Photoperiod (hrs dark / hrs light): 12/12 hrs
Route of administration:
oral: feed
Vehicle:
other: diet
Details on exposure:
VEHICLE
- Concentration in vehicle: LAS was added to their diets at levels of 0.02, 0.1 and 0.5%

Details on mating procedure
- Premating exposure period: 84 days (Po animals) and 80 – 85 days (F1b and F2b animals)
- Length of cohabitation: 17 days
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
2 years (3 generations)
Frequency of treatment:
Continuous in feed
Details on study schedule:
- F1 parental animals were not mated until 80-85 days old
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the Po animals in the study: 107-112 days old

Dose / conc.:
14 mg/kg bw/day
Remarks:
0.02% in diet
Dose / conc.:
70 mg/kg bw/day
Remarks:
0.1% in diet
Dose / conc.:
350 mg/kg bw/day
Remarks:
0.5% in diet
No. of animals per sex per dose:
20 males and 20 females per group
Control animals:
yes, concurrent no treatment
Details on study design:
Rats were divided into 4 groups and administered 0 (control), 0.02, 0.1 and 0.5% LAS added to their diets. Each group consisted of 50 males and 50 females randomized according to their litter and weight. After administration of LAS for 84 days when the parent animals (Po) were 107 – 112 days old, 20 female rats from each group were mated with 20 male rats from the same group. All females were separated from males on completion of seventeen days and placed in the opaque plastic litter boxes containing clean, dry saw dust and sufficient paper for nesting. Litters were examined for deformities and number of pups were counted. The first litters of F1a and F2a generation were sacrificed at 21 days of age and 10 days after sacrifice of litters, all females were remated with different males from same group to obtain the F1b generation. Twenty males and females of each group were selected at weaning to continue their respective diets and to be used for further reproduction studies and remaining weanling rats were examined and sacrificed. Reproduction studies on the F1b and F2b generations were started when the rats were 80 to 85 days old and were continued until the F3b generation was weaned. Average body weight, feed consumption and feed efficiency were recorded on weekly basis for the first 8 weeks for F1a and F2b generations. Once the reproductive studies were completed, five male and five female rats from each parenteral groups (F1a and F2b) were selected for necropsy and body weight, organ to body weight ratios were recorded. Routine hematology and histology studies were also performed. Weanling animals from F3a generation were treated similarly.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Gross examination of all animals along with routine hematology was performed after completion of reproductive study.

BODY WEIGHT: Body weight was recorded on weekly basis for first 8 weeks (F1b and F2b generation)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Feed consumption and feed efficiency were recorded on weekly basis for 12 weeks.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
Deformities and number of pups, average body weights, feed consumption, feed efficiency.

Postmortem examinations (parental animals):
Necropsy, body weight, organ to body weight ratios, routine hematology and histology
Postmortem examinations (offspring):
Necropsy, body weight, organ to body weight ratios, routine hematology and histology.
Reproductive indices:
Fertility, gestation, parturition, neonatal viability, lactation, and post-weaning growth
Offspring viability indices:
Body weight gain
Clinical signs:
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Mortality: There was no significant difference between mortality in control and treated group of rats as overall survival in this study exceeded 56% with the highest occurring in 350 mg/kg bw/day dose group (0.5% LAS in diet) as compared to 53% in control.

Body weight, organ weight and organ to body weight ratio: Body weight and organ to body weight ratios of selected animals from high level group and controls at 8, 15 and 24 months were within normal limits. A statistically significant decrease in liver weights was noted in male rats at the low and mid dose levels at the 8-month sacrifice. As the decreased liver weight was within normal range, was not seen at the highest dose level, nor was seen at the 15- and 24-month sacrifices, it was not considered biologically significant.

Food consumption and food efficiency: All groups were comparable in terms of food consumption and food efficiency.

Hematological findings: During routine hematological evaluations, statistically significant borderline values were scattered among various treatment groups and were not considered as treatment related.

Gross pathological and histopathological findings: No unusual lesions/abnormalities were noted upon gross examination of the visceral organs and there were no microscopic differences test and control animals as no test related response were observed in any of the tissues. Although higher incidences of incurrent degenerative diseases like chronic interstitial nephritis and adrenal telangiectasis and fibroadenomas were observed in final autopsy but these were not related to test material related exposure.

Reproductive performance: General reproduction including fertility, gestation, parturition, neonatal viability, lactation, and post-weaning growth were normal for all test groups and did not vary from controls.
Key result
Dose descriptor:
NOAEL
Effect level:
350 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
food efficiency
haematology
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
reproductive performance
Remarks on result:
other: No effects observed at the highest dose of 350 mg/kg bw/day(0.5% LAS in diet)
Critical effects observed:
not specified
Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
Mortality, body weight, organ to weight ratios, gross pathological and histopathological findings: These parameters were comparable to the control group for all three treatment groups.

Hematological parameters: In F1b female group receiving 70 mg/kg bw/day (0.1% LAS in diet) dose, red blood cell count was high but as it was within the normal range as determined by previous experiments conducted in the same laboratory.

Reproductive parameters: General reproductive parameters like fertility, gestation, parturition, neonatal viability, lactation and post weanling growth were normal across all test groups and comparable to control.
Key result
Dose descriptor:
NOAEL
Generation:
F1b
Effect level:
350 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
haematology
gross pathology
other: histopathology, organ / body weight ratios and reproductive parameters
Remarks on result:
other: No effects observed at the highest dose of 350 mg/kg bw/day (0.5% LAS in diet)
Critical effects observed:
not specified
Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
Mortality, body weight and organ to weight ratios: These parameters were comparable to the control group for all three treatment groups.

Hematological parameters: Red blood cell count was depressed in the F2b females from high dose test group as compared to control but was within the normal range as determined by previous experiments conducted in the same laboratory.

Gross pathological findings: No gross abnormalities were observed in any treatment group.

Histopathological findings: Pancreatic lesions consisting of acinar atrophy and tissue degeneration leading to the fibrous tissue replacement was observed in the F2b males and mild islet cell hyperplasia was also indicated. General lesions were also increased in the group receiving highest level of LAS. As similar lesions were also identified in the control males as well as control animals from the other studies, so these were not considered to be treatment related.

Reproductive parameters: General reproductive parameters like fertility, gestation, parturition, neonatal viability, lactation and post weanling growth were normal across all test groups and comparable to control.

Key result
Dose descriptor:
NOAEL
Generation:
F2b
Effect level:
350 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
haematology
gross pathology
other: histopathology, organ / body weight ratios and reproductive parameters
Remarks on result:
other: No effects observed at the highest dose of 350 mg/kg bw/day (0.5% LAS in diet)
Critical effects observed:
not specified
Key result
Reproductive effects observed:
no

F3b generation:

Rats sacrificed at weaning were normal w.r.t growth and organ to body weight ratios, gross pathology and histology and did not differs from control. Although statistically significant differences were observed were in hematological values between control and test groups, these differences were small and does not indicate a pattern as these animals were young leading to more individual variation as compared to mature animal.

NOAEL for F3b generation (based on absence of effects on growth and organ to body weight ratios, gross pathology and histology parameters at the highest tested dose): 350 mg/kg bw/day

Table 1: Average body weight gain and feed utilization (initial 12 weeks) for two year feeding study in rats (Buehler et. al., 1970)

Dietary level (%)

Dose (mg/kg bw/day)

Sex

Initial weight (g)

Final weight (g)

Weight gain (g)

Feed consumption (g)

Feed efficiency

0

0

M

59.5

491.1

431.6

2117.9

0.204

F

57.2

289.4

232.2

1579.9

0.147

0.02

14

M

59.4

485.1

425.7

2095

0.203

F

57

279

222

1545.7

0.144

0.1

70

M

59.9

492.8

432.9

2163.9

0.2

F

57.2

284.5

227.3

1568.5

0.145

0.5

350

M

59.8

480

420.2

2087.9

0.201

F

57.3

275.9

218.6

1520

0.144

Table 2: Growth and organ to body ratio's for two year feeding study in rats (Buehler et. al., 1970)

Time (Months)

Dietary level (%)

Dose (mg/kg bw/day)

Sex

Body weight (g)

Organ to body weight (expressed as % of total body weight)

Liver

Kidney

8

0

0

M

575

3.45

0.59

F

349

3.28

0.64

0.5

350

M

576

3.47

0.62

F

291

3.49

0.71

15

0

0

M

623

3.08

0.6

F

375

3.05

0.67

0.5

350

M

622

2.95

0.6

F

423

2.99

0.58

24

0

0

M

645

2.91

0.77

F

460

3.16

0.64

0.5

350

M

673

2.55

0.68

F

488

3.18

0.68

Table 3: Hematologic values for two year feeding study in rats (Buehler et. al., 1970)

Time (Months)

Dietary level (%)

Dose (mg/kg bw/day)

Sex

RBC count

Hemoglobin

Hematocrit

WBC count

Differential white cells

I

II

III

IV

V

8

0

0

M

6.81

15.5

48

7800

12

86

2

0

0

F

6.26

15

45

5700

9

89

1

0

1

0.5

350

M

5.81b

15.1

45

8900

11

85

1

0

3

F

5.7

14.6

42

4950

12

86

1

0

1

15

0

0

M

9.52

17.1

51

11100

10

84

5

0

1

F

6.88

16

44

6600

12

82

4

0

2

0.5

350

M

8.43

15.9

49

7600

13

83

3

0

1

F

7.74

14.7

45

6850

16

80

2

0

2

24

0

0

M

7.14

13.6

43

10500

23

72

4

0

1

F

6.65

13.2

43

10500

21

74

5

0

1

0.5

350

M

7.24

13.9

45

12600

35

59b

5

0

1

F

6.15

13.7

44

7900

25

68

6

0

1

b: Groups significantly different from control group

Differential white cells: Neutrophil / heterophil (I), Lymphocyte (II), Monocyte (III), Basophil (IV) and Eosinophil (V)

Table 4: Summary of mating for reproduction study in rats (Buehler et. al., 1970)                                                                                                                                                                 

Generation Dietary level (%) Dose (mg / kg bw/day) 0 day number in litter 5 days 21 days
No. in litter before culling  No. in litter after culling  Total weigh, origA Total weight after cullingA Number weaned  Body weights 
Male pups Female Pups MotherB
Number WeightA Number WeightA
F1a 0 0 12.3 12.1 7.81 129.2 87.7 7.4 3.6 201.3 4.1 205.3 319.5
0.5 350 12.7 12.5 7.6 123.4 81.6 7.5 3.8 197.6 3.7 178.3 324.8
F1b 0 0 12.4 11.5 7.6 120.2 82.4 6.4 3.5 197.9 2.9 157.8 363.1
0.5 350 13.7 12.9 8 128.4 83.9 7.6 3.5 183.5 3.7 185 355.9
F2a 0 0 12.2 11.7 7.9 145.5 102.4 7.7 3.4 158.9 4.4 189.2 316.2
0.5 350 12.7 12.1 7.9 141.7 96.4 7.6 4 183.1 3.6 154.2 323.2
F2b 0 0 11.8 11.6 7.5 141.4 94.9 7.5 3.7 209.5 3.8 208.4 350
0.5 350 12.2 11.8 7.8 130.1 89.3 7.6 4.2 201.4 3.4 166.5 352.4
F3a 0 0 12.6 12.3 8 136.7 91.9 7.9 4 216.8 3.9 202.6 328.7
0.5 350 11.4 11.2 7.8 127 90.7 7.8 4 202.7 3.8 186.2 316.8
F3b 0 0 12.9 12.3 7.9 151.2 102.6 8.2 3.8 213.3 4.3 233.5 356.1
0.5 350 12.1 11.7 7.7 150.1 100.6 7.7 4.3 240.1 3.4 181.4 337.6

                                   

A: Total group weight of pups (g)

B: Weight in grams

Table 5: Summary of hematology for two year reproduction study in rats (Buehler et. al., 1970)

Generation

Dietary level (%)

Dose (mg/kg bw/day)

Sex

RBC count

Hemoglobin

Hematocrit

WBC count

Differential white cells

I

II

III

IV

V

F1b

0

0

M

7.45

16.5

50

6800

14

80

3

0

3

F

6.03

16.9

49

5800

18

77

3

0

2

0.5

350

 

M

6.23

16.4

49

6800

16

80

2

0

2

F

5.88

16

47

6900

21

74

2

0

3

F2b

0

0

M

7.78

16.1

49

7900

13

79

6

0

2

F

7.27

16.2

48

5500

14

80

5

0

1

0.5

350

M

7.15

16

-

8400

10

85

4

0

1

F

6.15b

16.2

49

6400

11

83

5

0

1

b: Groups significantly different from control group

Differential white cells: Neutrophil / heterophil (I), Lymphocyte (II), Monocyte (III), Basophil (IV) and Eosinophil (V)

Conclusions:
Administration of linear alkylbenzene sulphonate sodium salt (Na-LAS) to male and female Charles river rats in their diet over 2 years across 3 generations at dose of 0, 14 (0.02%), 70 (0.1%) and 350 (0.5%) mg/kg bw/day resulted into NOAEL of 350 mg/kg bw/day for Po, F1, F2 and F3 generations (based on absence of marked effects on general reproduction parameters, growth, body weight, organ weight, body weight to organ weight ratio, hematology and histopathology parameters at the highest dose).

Executive summary:

The reproductive toxicity study of linear alkylbenzene sulphonate sodium salt (Na-LAS) in rats across three generations was conducted according to the accepted scientific principles.

 

Weanling rats and females after mated were housed in the individual wire bottom cages and opaque plastic boxes containing clean, dry sawdust and paper for nesting, respectively and maintained under standard laboratory conditions (temperature: 76 ± 3 °F, humidity: 50 ± 5 %, 12-hour light/12-hour dark cycle/day). The animals were allowed free access to the drinking water and food.

 

Male and female rats (Charles River strain) were used in this study. Experiment was initiated by randomizing rats into 4 groups of 50 males and 50 females each according to litter and weight and LAS was added into their diet at levels of 0 (control), 0.02, 0.1 and 0.5%. Dietary levels of 0.02, 0.1 and 0.5% corresponds to 14, 70 and 350 mg/kg bw/day, respectively. When parent animals (Po) were 107 – 112 days old and had received test diets for 84 days, groups of 20 female’s rats from each group were mated with 20 males from same group. After 17 days of initial exposure to males, females were transferred to the opaque plastic litter boxes with the clean and dry saw dust along with sufficient paper for nesting.

 

Litters were examined for deformities and number of pups were counted. On the fourth day, number of pups in each litter was limited to 8 for equalizing the stress of lactation among dams. The first litters of F1a generation were sacrificed at 21 days of age and after interval of 10 days, all females were remated with different males from the same group to obtain F1b generation. Twenty females and 20 males were selected from each group at weaning to continue their respective diets and used in further reproductive studies while and all remaining weanling rats were sacrificed and examined. These rats were housed in the individual wire bottom cages and fed the same diet. Reproduction studies on these rats were started at age of 80 - 85 days in a similar manner to obtain F2a and F2b generation and continued as such until F3b generation was weaned.

Average body weights, feed consumption and feed efficiency were recorded on weekly basis for first 8 weeks (F1b and F2b). After completion of the reproductive studies, 5 males and 5 female rats were selected from each parenteral group (F1b and F2b) for necropsy and body weight, organ to body weight ratio was recorded. Routine hematology and histology were also performed. Weanling animals from F3a generation were treated similarly. No significant effects were observed in the Po, F1b, F2b and F3b animals even at the highest dose i.e. 350 mg/kg bw/day (0.5% diet).

Based on above, administration of linear alkylbenzene sulfonate sodium salt to male and female rats by diet for three generation reproductive toxicity at dose of 0, 14 (0.02%), 70 (0.1%) and 350 (0.5%)  mg/kg bw/day resulted into NOAEL of 350 mg/kg bw/day for Po, F1, F2 and F3 generations (based on absence of marked effects on general reproduction parameters, growth, body weight, organ weight, body weight to organ weight ratio, hematology and histopathology parameters at the highest dose).

 

This reproductive toxicity study is acceptable and satisfies accepted scientific principles.

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
Food consumption was not determined between days 14 and 21 after parturition
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI (Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Germany
- Age at study initiation: (P) 16 days
- Weight at study initiation: (P) Males: 162.1 (142.5 – 186.5) g ; Females: 126.2 (110.6 – 145.1) g;
- Fasting period before study: none
- Housing: housed individually in type DK III stainless steel wire mesh cages
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland ad libitum
- Water: ad libitum
- Acclimation period: 16 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
The test substance (ethanolamine hydrochloride, EAH) was weighed and thoroughly mixed with a small amount of food. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer. Test diets were prepared at intervals, which guaranteed that the test substance in the diet remained stable throughout the feeding period.

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: over night
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged (how): individual
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of EAH in the diet over 32 days at room temperature was investigated analytically before the beginning of the study. Homogeneity and concentration control analyses were carried out at the beginning and toward the end of the premating periods. At least one analysis of test substance preparations for female animals was carried out during the gestation and lactation periods.

The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Positive control:
not done
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days and once daily on weekends


DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes
- Time schedule for examinations: body weights of F0 and F1 parents were determined once weekly; during gestation and lactation F0 and F1 females were weighed on days 0, 7, 14 and 20 of gestation, and on days 1, 4, 7, 14 and 21 after birth.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule: once weekly (over a period of at least 6 days each) and weekly during gestation (days 0-7, 7-14, 14-20 post coitum; p.c.) and lactation periods (days 1-4, 4-7, 7-14 post partum; p.p.).

OTHER:
The F1 and F2 pups were sexed on the day of birth (day 0 p.p.) and weighed on days 1, 4, 7, 14, and 21 p.p. Their viability was recorded. At necropsy, all pups were examined macroscopically (including weight determinations of brain, spleen and thymus in one pup/sex/litter).

Serum concentrations of the test substance:
Blood samples were taken from all F0 and F1 parental animals of each sex and test group during week 10 of premating treatment and the plasma was analyzed for the concentration of Ethanolamine hydrochloride
Oestrous cyclicity (parental animals):
Estrous cycle data were evaluated for F0 and F1 generation females over a three week period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.
Sperm parameters (parental animals):
Parameters examined in [all/P/F1] male parental generations:
motility, sperm head count, morphology
Postmortem examinations (parental animals):
All F0 and F1 parental animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention was given to the reproductive organs. The liver, kidneys, adrenal glands, testes, epididmides. Cauda epididymis, prostate, seminal vesicles, ovaries, uterus, spleen, brain, pituitary gland and thyroid glands (with parathyroids) were weighed and the vagina, cervix uterie, uterus, ovaries, oviducts, left testis, left epididymis, seminal vesicles, coagulation glands, prostate, pituitary gland, adrenal glands, liver, kidneys, spleen, brain, thyroids (with parathyroids)and all gross lesions were fixed in an appropriate fixative, histologically processed and examined by light microscopy. From both ovaries (”ovary 1” and “ovary 2”) of F1 female animals (control and top dose), five sections were taken from the proximal and the distal part of the ovaries, at least 100 µm apart from the inner third of the ovary. All ovarian sections were prepared and evaluated for numbers of primordial and growing follicles.
As soon as possible after termination, one portion of the liver (lobus medialis) of each 10 dams per group was sampled to be analyzed for choline concentration.
Postmortem examinations (offspring):
All pups with scheduled sacrifice (i.e. pups, which were culled on day 4 p.p., and pups, which were sacrificed on day 21 p.p. or subsequent days) were killed by means of CO2. The spleen and thymus of 1 pup/sex and litter from the F1 and F2 pups were weighed. All stillborn pups and all pups that died up to weaning were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.
Statistics:
see below
The following test substance-related effects/findings were recorded:

1000 mg/kg bw/d
F0 parental animal:

Yellow discolored urine for male and female parental animals
Statistically significantly decreased body weight gain of the dams during gestation, body weight 8% below control on gestation day 20
Statistically significantly decreased food consumption in parental females during lactation
Statistically significantly decreased sperm head count in the cauda epididymidis of males
Statistically significantly decreased absolute and relative weight of epididymides, cauda epididymidis and prostate in males
Statistically significantly less implantation sites
Statistically significantly increased post-implantation loss
Statistically significantly smaller litters


F1 parental animals:
Yellow discolored urine for male and female parental animals
Statistically significantly decreased body weight gain of the dams during gestation
Decreased food consumption in parental females during lactation
Statistically significantly decreased absolute and relative weight of epididymides and cauda epididymidis in males
Statistically significantly less implantation sites
Statistically significantly increased post-implantation loss
Statistically significantly smaller litters


300 mg/kg bw/d
F0 or F1 parental animals: No test substance-related adverse effects


100 mg/kg bw/d
F0 or F1 parental animals: No test substance-related adverse effects
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: fertility, reproductive performance and systemic toxicity
The following test substance-related effects/findings were recorded:

F1 or F2 pups: No test substance-related adverse effects


300 mg/kg bw/d
F1 or F2 pups: No test substance-related adverse effects


100 mg/kg bw/d
F1 or F2 pups: No test substance-related adverse effects
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no pre-and postnatal developmental toxicity
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no pre-and postnatal developmental toxicity
Reproductive effects observed:
not specified

Test substance stability:

The stability of test substance in rat diet was demonstrated for a period of 32 days at room temperature in a different batch of comparable quality, which was not used for the study. The homogeneity of the mixtures was verified. The concentration control analyses of the samples taken revealed that the values were within a range of 90-110% of the nominal concentration in all analyses at all time points, with the exception of one concentration in the feed of the high-dose group (88%).

Plasma concentrations of 2 -aminoethanol were below 3 mg/kg for all control animals, <3 - 4 mg/kg for the low dose animals, 8 - 11 mg/kg for the mid dose animals and 60 – 81 mg/kg for the high dose animals.

Toxicokinetic data of 2 -aminoethanol (calculated as 2 -aminoethanol hydrochloride) from this two-generation reproduction toxicity study show a dose dependency of the plasma levels of 2 -aminoethanol in the experimental animals and there with prove the bioavailability of 2 -aminoethanol hydrochloride in principle.

 

Under these conditions, no test substance-related findings from clinical examinations or gross and histopathology were observed, which indicate that the administration of the test compound via the diet adversely affected the fertility or reproductive performance of the F0 or F1 parental animals up to and including a nominal dose of 300 mg/kg bw/d. Estrous cycle data, mating behavior, conception, gestation, parturition, lactation and weaning as well as sperm parameters, sexual organ weights and gross and histopathological findings of these organs (including differential ovarian follicle counts in the F1 females) were comparable between the rats of all test groups.

At the high-dose level (1000 mg/kg bw/d), absolute and relative weights of epididymides and cauda epididymidis were decreased and, in the F0 generation only, the number of homogenization resistant caudal epididymal sperm was slightly, but significantly reduced. However, histomorphological correlates for these findings were missing.

 

In the high-dose F0 and F1 generation females (1000 mg/kg bw/d), decreased numbers of implants and increased resorption rates resulted in significantly smaller litters, giving evidence for an adverse effect of the test compound on fertility and/or reproductive performance at high doses. It has to be noted that a dose of 1000 mg/kg bw/d also caused beginning systemic toxicity in these females, as was indicated by reduced food consumption and/or body weight gain during gestation/lactation.

 

All data recorded during gestation and lactation in terms of embryo-/fetal and pup development gave no indications for any developmental toxicity in the F1 and F2 offspring up to a dose level of 1000 mg/kg bw/d. The test substance did not adversely influence pup viability, body weight, sex ratio and sexual maturation.

 

Thus, under the conditions of the present two-generation reproduction toxicity study, the NOAEL (no observed adverse effect level) for fertility, reproductive performance and systemic toxicity in parental F0 and F1 Wistar rats is 300 mg/kg bw/d.

 

The NOAEL for pre-and postnatal developmental toxicity in their offspring is 1000 mg/kg bw/d.

Tables

Mean test substance intake (mg/kg bw/d; minimum value / maximum value)

 

Test group 01
(100 mg/kg bw/d)

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

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

F0 males

94.3 (72.4 / 102.5)

283.2 (218.4 / 309.4)

943.3 (716.7 / 1032.6)

F0 females (premating)

96.7 (80.5 / 100.7)

289.6 (241.2 / 304.9)

964.4 (792.4 / 1017.8)

F0 females
(F1 litter)
- gestation period
- lactation period*



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



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



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

* = Days 1–14 p.p. only

Absolute organ weights (P-generation)

Compared to the controls (= 100%), the following values (in %) were significantly changed (printed in bold):

 

Male animals

Female animals

Group

01

100 mg/kg bw/d

02

300 mg/kg bw/d

03

1000 mg/kg bw/d

01

100 mg/kg bw/d

02

300 mg/kg bw/d

03

1000 mg/kg bw/d

Brain

99%

100%

97%*

 

 

 

Cauda epididymis

99%

102%

88%**

 

 

 

Epididymides

100%

101%

92%**

 

 

 

Prostate

92%

99%

86%**

 

 

 

Spleen

 

 

 

105%*

107%

97%

 

*: p≤0.05; **: p≤0.01

 

All other mean absolute weight parameters did not show significant differences compared to the control groups.

 

The decrease of absolute weights of cauda epididymis, epididymides, and prostate in male top-dose animals (1000 mg/kg bw/d) were considered as treatment-related effects.

 

The decrease of brain weights in top-dose males (1000 mg/kg bw/d) as well as the increase of spleen weights in low-dose females (100 mg/kg bw/d) was considered as incidental and not treatment-related due to a missing dose-response relationship.

Absolute organ weights (F1 generation)

Compared to the controls (= 100%), the following values (in %)were significantly changed (printed in bold):

 

 

Male animals

Female animals

Group

11

100 mg/kg bw/d

12

300 mg/kg bw/d

13

1000 mg/kg bw day

11

100 mg/kg bw/d

12

300 mg/kg bw/d

13

1000 mg/kg bw/d

Cauda epididymis

96%

99%

88%**

 

 

 

Epididymides

100%

101%

91%**

 

 

 

Kidneys

99%

106%*

111%**

103%

106%**

115%**

Spleen

99%

103%

92%*

 

 

 

Thyroid glands

106%

99%

109%*

110%

118%**

111%*

 

*: p≤0.05; **: p≤0.01

All other mean absolute weight parameters did not show significant differences compared to the control groups.

The decrease of absolute weights of cauda epididymis and epididymides in male top-dose animals (1000 mg/kg bw/d) were considered to be treatment-related.

 

The increase of absolute kidney weights of male and female animals in mid- (300 mg/kg bw/d) and top-dose (1000 mg/kg bw/d) groups, respectively, was statistically significant. Because no histomorphological correlate was detected, a treatment-related weight increase was less likely.

 

The decrease of spleen weights in top-dose males as well as the increase of thyroid glands in top-dose males and mid- and top-dose females, respectively, is considered incidental and not treatment-related due to a missing dose-response relationship.

Endpoint:
fertility, other
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1975
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
A composite of dyes and base components found in semi-permanent hair color products (containing monoethanolamine at 22.42%) was administered in the diet to rats to assess the impact of formulation on teratogenic and reproductive performance of animals.

Male and female Sprague-dawley CD strain rats were exposed to 0, 1950, and 7800 ppm of composite in diet. The study was conducted in two parts. In Part I (male fertility study), the females received the basal diet from 8 week prior to mating through the weaning of their litters. The males siring these litters were fed the test diets for 8 weeks prior to mating and during the mating period. In Part II (female fertility study), males received the basal diet for 8 weeks prior to and during mating, while the females received the test diets 8 week prior to mating and during gestation and 21 days of lactation. Pregnant females (one female pregnant by each male) were euthanized at day 13 of pregnancy for obtaining information for the early stages of gestation. The uterus was examined for the number and distribution of embryos, the presence of empty implantation sites, and the number of embryos undergoing resorption. The remaining dams were allowed to deliver normally. A necropsy was performed on all females that did not deliver a litter to determine whether pregnancy had occurred. The duration of gestation was noted, and the litters were examined for numbers of live and stillborn pups and gross abnormalities. The pups were weighed at birth, and at 4 and 21 days. At 21 days all surviving pups were euthanized and examined grossly for abnormalities.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague-Dawley CD strain rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts.
- Weight at study initiation:
(P) Male rats- 240-280 g and female rats- 180-220 g
- Fasting period before study: Not specified
- Diet: Basal diet of Purina laboratory chow; ad libitum
- Water: ad libitum
- Acclimation period: Not specified
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
- DIET PREPARATION
- Rate of preparation of diet: Diets were prepared twice weekly
Details on mating procedure:
- M/F ratio per cage: 1 male: 2 females
- Length of cohabitation: Overnight, from 4 pm to 8 am.
- Proof of pregnancy: Sperm in vaginal smear was referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: No. A female was considered infertile if she failed to become pregnant after mating with two different males for 10 days each.
- After successful mating each pregnant female was caged individually
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Females:8 weeks prior to mating and during gestation and 21 days of lactation (part II).
Males: 8 weeks prior to mating and during the mating period (part I)
Frequency of treatment:
Daily (exception: food was withdrawn for the time of mating)
Details on study schedule:
The study was divided into two parts:
In Part I, the females received the basal diet from 8 week prior to mating through the weaning of their litters. The males siring these litters were fed the test diets for 8 weeks prior to mating and during the mating period.

In Part II, males received the basal diet for 8 weeks prior to and during mating, while the females received the test diets 8 week prior to mating and during gestation and 21 days of lactation.
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
1 950 ppm (nominal)
Remarks:
corresponds to 18 and 26 mg/kg bw/day for females and males respectively
Dose / conc.:
7 800 ppm (nominal)
Remarks:
corresponds to 74 and 116 mg/kg bw/day for females and males respectively
No. of animals per sex per dose:
10 males and 20 females per dose level
Control animals:
yes, plain diet
Positive control:
No positive control was included in the study
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: YES

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): YES
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED: The duration of gestation was noted and the litters’ were examined for numbers of live and stillborn pups and gross abnormalities. The pups were weighed at birth, and at 4 and 21 days. At 21 days, all surviving pups were euthanized and examined grossly for abnormalities.

GROSS EXAMINATION OF DEAD PUPS: Yes for gross abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not examined

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not examined
Postmortem examinations (parental animals):
SACRIFICE AND GROSS NECROPSY
- Male animals: Not specified
- Maternal animals: One female pregnant by each male was euthanized on day 13 of her pregnancy to obtain information regarding the early stages of gestation. The uterus was examined for the number and distribution of embryos, the presence of empty implantation sites, and the number of embryos undergoing resorption. Each embryo was examined under a dissecting microscope. The remaining dams were delivered normally. A necropsy was performed on all females that did not deliver a litter to determine whether pregnancy had occurred

HISTOPATHOLOGY / ORGAN WEIGHTS: Not examined
Postmortem examinations (offspring):
SACRIFICE AND GROSS NECROPSY
For both male and female fertility (part I and II) studies, the F1 surviving pups were euthanized and examined grossly for abnormalities at day 21.

HISTOPATHOLOGY / ORGAN WEIGTHS: Not examined
Statistics:
The data were subjected to statistical analysis, using the 95% confidence level. The methods used included chi square test, analysis of variance and t test, and the Fisher exact probability test

Reproductive indices:
Period of gestation
Female fertility index: Number of pregnant females/ Number of females mated as percent.
Male fertility index: Number of pregnant females siring litters/ Number mated to fertile females as percent.
Gestation index: Percent of pregnancies resulting in litters cast alive.
Offspring viability indices:
Viability index: Percent of pups cast alive that survived at 4 days.
Lactation index: Percent of pups alive at 4 days that survived to weaning at 21 days.
Clinical signs:
no effects observed
Mortality:
not specified
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
There were no dose-related significant differences in any of the parameters examined which included male and female fertility, length of gestation and numbers of females with resorption sites. The female fertility index in the high dosage group in Part I and the average pup weight in the high dosage group in Part II were lower than the control values, but the differences were not statistically significant at the 95% confidence level. There were no effects on food consumption and body weight gains of either males or females.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 7 800 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
water consumption and compound intake
organ weights and organ / body weight ratios
reproductive performance
Remarks on result:
other: No adverse effect was observed on clinical signs, mortality, body weight, food consumption, organ weights and reproductive performance. Based on food consumption, this NOAEL corresponds to 74 mg/kg bw/day in males and 116 mg/kg bw/day in females.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined
There were no dose-related significant differences in any of the parameters examined which included live pups per litter, pup body weights, and pup survival. No abnormal pups were seen upon dissection of embryos after 13 days of gestation or upon gross examination at weaning after 21 days.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 7 800 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality
body weight and weight gain
other: Lactation index
Remarks on result:
other: No adverse effect was observed on pups per litter, pup body weights, and pup survival.

No dose-related significant differences in any of the parameters examined which included male and female fertility, length of gestation, numbers of females with resorption sites, live pups per litter, pup body weights, and pup survival were observed. The female fertility index in the high dosage group in Part I and the average pup weight in the high dosage group in Part II were lower than the control values, but the differences were not statistically significant at the 95%, confidence level. At the dietary concentrations fed, there were no effects on food consumption and body weight gains of either males or females. No abnormal pups were seen upon dissection of embryos after 13 days of gestation or upon gross examination at weaning after 21 days.

Table 1: Effect of dye/composite on fertility and general reproductive /performance of male rats (Wernick et al., 1975)

  Concentration in Diet (%)
  0 0.195 0.78
Summary of mating performance
mg/kg bw/day (average) 0 86 351
No. of males 10 10 10
No. of females 20 20 20
No. of pregnancies 17 19 13
Female fertility indexa,b 85 95 65
No. unproductive index 1 0 2
Male fertility indexa,c 90 100 8
Summary of treated male induced pregnancies terminated on the 13th day of gestation
mg/kg bw/day (average) 0 86 351
No. pregnant females sacrificed 8 9 8
Average no. of embryos  
Left horn 5.3 6 5.7
Right horn 6.6 6.1 6.3
Total 11.9 12.1 12
No. empty implant sites 0 0 0
No. undergoing resorption 7 5 6
No. abnormalities 0 0 0
No. litters with resorptiond 5 5 4
Summary of treated male induced pregnancies allowed to litter normally
mg/kg bw/day (average) 0 86 351
No. of females 9 10 5
Average length of gestation (days)d 21.6 21.7 21.4
Gestation indexe,f 100 100 100
Average no. of pups per littere 10.5 11.1 10.8
Percent alive at: birth 94 98 98
4 days 92 98 94
21 days 89 98 93
Viability indexe,g 98 99 96
Lactation indexe,h 98 100 98
Average body weight of pups at: birthe 6.5 6 6.4
4 dayse 10.6 9.3 9.7
21 dayse 46.8 48.1 48.2

a Statistical analysis by fischer probability test on male and female fertility indices.

b Female fertility index: No. of pregnant females/no. of females mated as percent.

c Male fertility index: No. of pregnant females siring litters/no. mated to fertile females as percent.

d Statistical analysis by method of chi-square on number of litters with resorption sites.

e Statistical analysis by analysis of variance on average body weight of pups at birth and at 4 and 21 days, length of gestation, and number of pups per litter.

f Gestation index = percent of pregnancies resulting in litters cast alive.

g Viability index = percent of pups cast alive that survived at 4 days.

h Lactation index = percent of pups alive at 4 days that survived to weaning at 21 days.

Table 2: Effect of dye/composite on fertility and general reproductive /performance of female rats (Wernick et al., 1975)

  Concentration in Diet (%)
  0 0.195 0.78
Summary of mating performance
mg/kg bw/day (average) 0 124 554
No. of females 20 20 20
No. of males 10 10 10
No. of pregnancies 19 18 19
Female fertility indexa 95 90 95
No. unproductive index 0 0 0
Summary of treated male induced pregnancies terminated on the 13th day of gestation
No. pregnant females sacrificed 9 8 9
Average no. of embryos  
Left horn 6.2 6.3 7.3
Right horn 6 5.4 5.8
Total 12.2 11.7 13.1
No. empty implant sites 1 0 0
No. undergoing resorption 3 2 6
No. abnormalities 0 0 0
No. litters with resorptionb 3 2 4
Summary of treated male induced pregnancies allowed to litter normally
mg/kg bw/day (average) 0 124 554
No. of females 10 10 10
Average length of gestation (days)c 21.7 21.4 21.1
Gestation index 100 100 100
Average no. of pups per litterc 10.5 11.6 11.6
Percent alive at: birth 95 91 96
4 days 88 78 88
21 days 87 77 86
Viability index 92 86 92
Lactation index 99 98 98
Average body weight of pups at: birthc 6.5 6.3 6.1
4 daysc 10.1 9.6 9.6
21 daysc 47.1 49.3 44.3

a Statistical analysis by fischer probability test on male and female fertility indices.

b Statistical analysis by method of chi-square on number of litters with resorption sites.

c Statistical analysis by analysis of variance on average length of gestation, number of pups per litter and body weight at birth and at 4 and 21days.

Table 3: Dye/base composite (Wernick et al., 1975)

CTFA dictionary name Dye
Common name Chemical abstract name %
Acid Orange 3 C.I. Acid Orange 3 Sodium 4-anilino-2’,4’-dinitrodiphenylamine- 3-sulfonate 0.24
H.C. Blue No. 2 N1, N4, N4-(2-Hydroxyethyl)-2-nitrop- phenylenediamine 2,2’-[4-[(2-Hydroxyethylamino)-3- nitrophenyl]imino]diethanol 1.63
Celliton Fast Navy Blue BRAa     0.64
2-Nitro-p-phenylenediamine 2-Nitro-p-phenylenediamine 1,4-Diamino-2nitrobenzene 0.24
4-Nitro-o-phenylenediamine 4-Nitro-o-phenylenediamine 1,2-Diamino4nitrobenzene 0.16
2-Amino-4-nitrophenol 2-Amino-4nitrophenol 2-Amino-4-nitrophenol 0.05
H.C. Yellow No. 5 N1-(2-Hydroxyethyl)-4nitro-o-phenylenediamine 2-[2-Amino-4-nitroanilinol]ethanol 0.05
H.C. Yellow No. 4 N, N-bis(2-Hydroxyethyl)-2-amino- 5-nitrophenol 2,2’[(2-Hydroxy4nitrophenyl)-imino]-diethanol 0.31
Disperse Violet 11 C.I. Disperse Violet 11 Anthraquinone 0.4
Disperse Blue 1 C.I. Disperse Blue 1   0.61
Disperse Black 9 C.I. Disperse Black 9 -Diazo 0.13
H.C. Blue No. 1 N4, N4-bis-(2-Hydroxyethyl)-N l-methyl- 2-nitro-p-phenylenediamine 2,2’-[[4-(Methylamino)-3-nitrophenyl]-imino]diethanol 1.54
H.C. Red No. 3 Nr-(2-Hydroxyethyl)-2-nitro-p-phenylenediamine 2-[4-Amino-2-nitroanilino]-ethanol 0.02
H.C. Yellow No. 3 N1-tris (Hydroxymethyl)-methyl-4-nitro-o- phenylenediamine 2-bis (Hydroxymethyl)-2-[2-amino-4-nitroanilino]ethanol 0.65
H.C. Yellow No. 2 N-(2-Hydroxyethyl)-2-nitroaniline 2-[2-Nitroanilino]-ethanol 0.28
Base
Lauramide DEA 20.22
Imino-bis propylamine 9.64
cellulose ether 17.94
Citric acid 16.02
BHT 1.61
Sodium m-nitrobenzene sulfonate 2.25
TEA-Dodecylbenzene sulfonate 1.61
Monoethanolamine 22.42
Perfume 0.67
Perfume 0.67
  100

a: A mixture of: Disperse Yellow 1-2,4-Dinitro-4’-hydroxy diphenylamine.

Disperse Blue 1-1,4,5,8-Tetraamino anthraquinone.

Disperse Violet 4-1-Amino+methylamino anthraquinone.

Disperse Red 17-4-(bis-hydroxyethyl) amino-2-methyl-4’-nitroazobenzene.

Conclusions:
Administration of composite (containing 22.42% monoethanolamine) to male and female Sprague Dawley rats at dose levels of 0, 1950, and 7800 ppm [equivalent to 0, 86 and 351 mg/kg bw/day in Part I (male fertility study) and 0, 124 and 554 mg/kg bw/day (female fertility study)] resulted in no dose-related significant differences in any of the parameters examined which included male and female fertility, length of gestation, numbers of females with resorption sites, live pups per litter, pup body weights, and pup survival compared to controls. Therefore, composite (containing 22.42% monoethanolamine) has no impact on male and female fertility up to 7800 ppm (= 1748.76 ppm a.i. monoethanolamine).
Executive summary:

The toxicity to reproduction of monoethanolamine was conducted in Sprague Dawley CD strain rats. A composite of dyes and base components found in semi-permanent hair color products containing 22.42% of monoethanolamine was administered in the diet to rats.

Male and female Sprague-dawley CD strain rats were used in the study. body weights of the male rats ranged from 240 to 280 g while those of the females ranged from 180 to 220 g. A composite (containing 22.42% monoethanolamine) was incorporated into the basal diet at concentrations to give dosages of 0, 1950, and 7800 ppm. Diets were prepared fresh twice weekly.

The study was conducted in two parts. In Part I (male fertility study), the females received the basal diet from 8 week prior to mating through the weaning of their litters. The males siring these litters were fed the test diets for 8 weeks prior to mating and during the mating period. In Part II (female fertility study), males received the basal diet for 8 weeks prior to and during mating, while the females received the test diets 8 week prior to mating and during gestation and 21 days of lactation.

One male and two female rats was cohabitated together for mating. The day on which sperm were detected in vaginal smear was detected was designated as Day 0 of pregnancy (GD 0). One female pregnant by each male was euthanized by chloroform inhalation on day 13 of her pregnancy to obtain information regarding the early stages of gestation. The uterus was examined for the number and distribution of embryos, the presence of empty implantation sites, and the number of embryos undergoing resorption. Each embryo was examined under a dissecting microscope. The remaining dams were delivered normally.

A necropsy was performed on all females that did not deliver a litter to determine whether pregnancy had occurred. The duration of gestation was noted, and the litters were examined for numbers of live and stillborn pups and gross abnormalities. The pups were weighed at birth, and at 4 and 21 days. At 21 days all surviving pups were killed by chloroform inhalation and examined grossly for abnormalities.

No dose-related significant differences in any of the parameters examined which included male and female fertility, length of gestation, numbers of females with resorption sites, live pups per litter, pup body weights, and pup survival were observed. The female fertility index in the high dosage group in Part I and the average pup weight in the high dosage group in Part II were lower than the control values, but the differences were not statistically significant at the 95%, confidence level. At the dietary concentrations fed, there were no effects on food consumption and body weight gains of either males or females. No abnormal pups were seen upon dissection of embryos after 13 days of gestation or upon gross examination at weaning after 21 days.

Based on above, administration of composite (containing 22.42% monoethanolamine) to male and female Sprague Dawley rats at dose levels of 0, 1950, and 7800 ppm [equivalent to 0, 86 and 351 mg/kg bw/day in Part I (male fertility study) and 0, 124 and 554 mg/kg bw/day (female fertility study)] resulted in no dose-related significant differences in any of the parameters examined which included male and female fertility, length of gestation, numbers of females with resorption sites, live pups per litter, pup body weights, and pup survival compared to controls. Therefore, composite (containing 22.42% monoethanolamine) has no impact on male and female fertility up to 7800 ppm (= 1748.76 ppm a.i. monoethanolamine).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
350 mg/kg bw/day
Study duration:
chronic
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

Reproductive toxicity 

  

 Study 1 (C10-14LAS-Na): 

  

A three-generation reproductive toxicity study was conducted with the read-across substance C10-14LAS, sodium salt in Charles River rats. The test substance was administered to groups of 50 males and 50 females in the feed at doses of 0, 0.02, 0.1, and 0.5% (0, 14, 70, and 350 mg/kg bw/day) for 84 days and evaluated for three generations (a total of 2 years). When the P0-generation was 107-112 days old, 20 females from each dose group were mated with 20 males from the same group and maintained together for 17 days. The first litters of each generation (Fla- and F2a-generations) were sacrificed at 21 days of age. Ten days after the final litter was sacrificed, all females were re-mated with different males from the same group to obtain the F1b-generation. From the Flb-generation, 20 males and females of each group were selected at weaning to continue their respective diets and to be used for further reproduction studies. Reproduction studies on the F1b and F2b-generations were started when the rats were 80 to 85 days old and were continued until the F3b-generation was weaned. All rats sacrificed at weaning were normal with respect to growth, organ to body weight ratios, gross pathology, and histopathology, and did not vary from controls. There were a number of statistically significant hematologic values, though these differences were small and did not indicate a trend or pattern. Overall, no significant effects were observed up to the highest dose tested. Based on the results, the systemic, reproductive and developmental toxicity NOAEL of 350 mg/kg bw/day for P0, F1, F2 and F3 generations based on the absence of treatment-related effects on general reproduction parameters, growth, body weight, organ weight, body weight to organ weight ratio, haematology and histopathology up to the highest dose (Buehler, 1971).

  

 Study 2 (MEA): 

 

A two-generation reproductive toxicity study was conducted with monoethanolamine (MEA) according to OECD Guideline 416 in Crl:WI male and female rats. The test substance was administered to groups of 25 male and 25 female rats in feed at doses of 0, 100, 300 and 1000 mg/kg bw/day. There were no treatment-related effects on clinical examinations, gross pathology and histopathology of the F0 or F1 parental animals up to the dose of 300 mg/kg bw/day. Oestrous cycle data, mating behaviour, conception, gestation, parturition, lactation and weaning as well as sperm parameters, sexual organ weights and gross and histopathological findings of these organs (including differential ovarian follicle counts in the F1 females) were comparable between the treated and control group rats. At the high dose, absolute and relative weights of epididymis and cauda epididymis were decreased and, in the F0 generation only, the number of homogenisation-resistant cauda epididymal sperm was slightly, but significantly reduced. However, there were no histomorphological correlation for these findings. In the high dose F0 and F1 generation females, decreased numbers of implants and increased resorption rates resulted in significantly smaller litters. This was probably due to systemic toxicity in these females, as indicated by reduced food consumption and/or body weight gain during gestation/lactation. All data recorded during gestation and lactation in terms of embryo/foetal and pup development gave no indications for any developmental toxicity in the F1 and F2 offsprings up to the highest dose level. Based on the findings, the systemic/reproductive toxicity NOAEL in the parental animals was determined to be 300 mg/kg bw/day and the developmental toxicity NOAEL in the F1 and F2 generations was determined to be 1000 mg/kg bw/day (ACC and CEFIC, 2009).

  

Study 3 (MEA): 

  

A reproductive toxicity study was conducted with monoethanolamine (MEA) conducted in Sprague Dawley CD strain rats. The protocol for this study closely matches that of a reproductive/developmental screening study. The test substance was administered in the diet (in the form of a composite dye containing 22.42% of MEA) at doses of 0, 1950, and 7800 ppm. The study was conducted in two parts. In Part I (male fertility study), the females received the basal diet from 8 week prior to mating through the weaning of their litters. The males siring these litters were fed the test diets for 8 weeks prior to mating and during the mating period. In Part II (female fertility study), males received the basal diet for 8 weeks prior to and during mating, while the females received the test diets 8 week prior to mating and during gestation and 21 days of lactation. No treatment-related significant differences in any of the parameters examined which included male and female fertility, length of gestation, numbers of females with resorption sites, live pups per litter, pup body weights, and pup survival were observed. The female fertility index in the high dose group in Part I and the average pup weight in the high dose group in Part II were lower than the control values, but the differences were not statistically significant. There were no adverse effects on food consumption and body weight gain of either sex. No abnormal pups were seen upon dissection of embryos after 13 days of gestation or upon gross examination at weaning after 21 days. Under the study conditions, the reproductive toxicity NOAEL was determined to be 7800 ppm (1748.76 ppm a.i. MEA; based on food consumption, this NOAEL corresponds to 74 mg/kg bw/day in males and 116 mg/kg bw/day in females) based on absence of dose-related significant differences in any of the parameters examined which included male and female fertility, length of gestation, numbers of females with resorption sites, live pups per litter, pup body weights, and pup survival compared to controls (Wernick, 1975).

Effects on developmental toxicity

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of this study was to evaluate the teratogenic potential of linear alkylbenzene sulphonate (LAS) in rats. Twenty female rats/dose were divided into 5 groups and administered 0 (water), 0.2, 2.0, 300 and 600 mg/kg bw/day of test substance by oral gavage from day 6 to 15 of gestation. Throughout experiment animals were observed daily for signs of malreaction and weighed regularly. After euthanasia on day 20, their uterine content was examined for number of implantations, viable young and embryonic deaths along with ovaries, number of corpora lutea and viable pups. Body weight, incidents of external malformation, visceral and skeletal anomalies were also recorded in pups. Non parametric methods were used for statistical analysis and on the basis of adverse effects, NOAEL value was derived for maternal toxicity and teratogenicity.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: CD rats
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass., U.S.A. and St-Aubin-les-Elbeuf, France.
- Housing: Female rats (5 per cage) were housed in opaque plastic cages.
- Diet: Spratt’s Laboratory Diet No. 1, ad libitum.
- Drinking water: ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 1°C
- Humidity: 50 ± 5 % RH
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- Preparation of dosing solutions: Doses were prepared on daily basis as series of graded aqueous solutions for oral dosing.
- Administration of dosage: Standard volume of doses was administered orally by gastric intubation. Control animals were dosed with water.
- Duration of dosage: Doses were commenced on day 6 after detection of vaginal plug in rats and continued daily up to and including day 15 of gestation.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Mating was confirmed by the detection of vaginal plug in rats.
Duration of treatment / exposure:
Animals were dosed for 10 days [from Day 6 (after detection of vaginal plug) to Day 15].
Frequency of treatment:
daily from day 6 after detection of the vaginal plug to till day 15
Duration of test:
21 days
Dose / conc.:
0 mg/kg bw/day
Remarks:
(control)
Dose / conc.:
0.2 mg/kg bw/day
Dose / conc.:
2 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
600 mg/kg bw/day
No. of animals per sex per dose:
20 female rats per dose
Control animals:
yes
Details on study design:
- Dose selection rationale: Two doses formed the basis for safety evaluation (0.2 and 2.0 mg/kg bw/day) because the likely maximum human intake of detergent from ordinary kitchen use has been estimated at 0.14 mg/kg bw/day, thus providing factors of 1-2 times the human exposure level. Two further doses (300 and 600 mg/kg bw/day) were also investigated based on previous toxicity data suggesting that these would impair maternal economy and result in obvious adverse effects.
Maternal examinations:
All animals were observed daily for signs of malreaction and were weighed regularly throughout gestation. All animals that died, and survivors at termination, were dissected and examined for macroscopic changes.
Ovaries and uterine content:
After euthanasia, uteri were dissected and examined to determine the number of:
a) Implants
b) Viable young
c) Embryonic deaths (abortion/resorption sites)

Ovaries were examined and number of corpora lutea were also recorded.
Fetal examinations:
After weighing, fetus were examined for external malformations, visceral anomalies by free and sectioning and skeletal examination was performed by alizarin staining.
Statistics:
Differences in mean values were statistically analyzed by non-parametric method i.e. Wilcoxon test.
Historical control data:
In CD rats, percentage of major malformations, minor visceral anomalies and minor skeletal anomalies were recorded as 0.41%, 2.02% and 2.35%, respectively in 51349 examined fetuses.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Toxic effects were generally associated with the gastrointestinal tract disturbances.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Single mortality was observed at 600 mg/kg bw/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Retarded weight gain was observed on 600 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
Retarded weight gain was observed on 600 mg/kg bw/day. No other significant maternal toxicity was observed at all doses when compared to the control group. Mating index was also comparable across all doses when compared to the control group.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Other effects:
not specified
Details on maternal toxic effects:
Body weight: Moderate maternal toxicity was observed in 600 mg/kg bw/day on the basis of retarded weight gain.

Litter loss: Only single litter was lost at 0.2 mg/kg bw/day but not significant as no dose response relationship was followed.

Pre-implantation embryonic losses were 18.1%, 23.2%, 36.9%, 15.6% and 20.3% at 0, 0.2, 2, 300 and 600 mg/kg bw/day, respectively.

Post-implantation embryonic losses were 2.8%, 12.4%, 5.9%, 3.6% and 3.1% at 0, 0.2, 2, 300 and 600 mg/kg bw/day, respectively.

Embryonic deaths were 0.3, 0.7, 0.6, 0.4 and 0.4 at 0, 0.2, 2, 300 and 600 mg/kg bw/day, respectively.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: mortality and retarded weight gain at 600 mg/kg bw/day
Abnormalities:
not examined
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
EMBRYO TOXICITY
Fetal weights: Although the fetal weights differed significantly at 0.2 and 2 mg/kg bw/day, these changes does not represent an adverse change because there was no consistent dose relationship and were invariably due to higher mean pub weight in the test group.

Fetal loss: No test substance related effect or statistically significant differences were observed.

Litter size and weight: Litter size and weights were comparable across all doses when compared to the control group.

MALFORMATIONS:
No test substance related or statistically differences in the incidence of major malformations observed even a maternally toxic dosage but minor visceral anomalies were increased at 600 mg/kg bw/day (not significant statistically) but same incident was also observed in control group.

DEVELOPMENTAL VARIATIONS:
Incidences of skeletal abnormalities were comparable across all doses when compared to the control group.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: marginal retardation of sternebral ossification at the highest dose
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
Treatment related:
not specified
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects

Formula for calculation of mating index:

Mating index: (number of confirmed mating/ number of animals cohabitated) X 100

Table 1: Mating index of the maternal animals (Palmer et.al., 1975)

Dose 

Number of confirmed mating

Number of animals cohabitated

 
Mating index %  
0 15 20 75
0.2 15 20 75
2 18 20 90
300 16 20 80
600 16 19 84

Table 2: Summary of adult performance (Palmer et.al., 1975)

Observations  Number of animals at dosage (mg/kg bw/day)
0 0.2 2 300 600
Mated  20 20 20 20 20
Died  0 0 0 0 1
Non-pregnant 5 5 2 4 3
Total litter loss 0 1 0 0 0
With viable young  15 14 18 16 16
Body weight change  - - - - retarded gain 

Table 3: Group mean litter data (Palmer et.al., 1975)

Dosage (mg/kg bw/day)

Number of litters  Litter size viable young  Embryogenic deaths  Implantations  Corpora lutea  Embryonic loss %  Litter weight (g) Fetal weight (g)
Pre-implantation  Post-implantation 
0 15 (A & B) 9.9 0.3 10.3 12.5 18.1 2.8 36.15 3.66
0.2 15 (A) 9.1 0.7 9.8 12.7 23.2 12.4 - -
14 (B) 9.8 0.6 10.4 12.8 18.4 6.1 37.14 3.84a
2 18 (A & B) 8.2 0.6 8.8 13.9 36.9 5.9 31.62 3.94b
300 16 (A & B) 9.6 0.4 10 12.4 15.6 3.6 35.72 3.78
600 16 (A & B) 10.4 0.4 10.8 13.9 20.3 3.1 37.49 3.63

A: Mean value includes all surviving animals showing evidence of implantation, including those with total litter loss

B: Mean value includes only animals bearing viable young at termination

Difference from controls statistically significant at Wilcoxon test: aP < 0.05 and bP < 0.001.

Table 4:

a)       Group mean incidence for major and minor malformations (Palmer et.al., 1975)

Dosage (mg/kg bw/day) Number of young
Major malformations  Minor malformations*
Gross or visceral Skeletal
Examined  Affected  Examined  Affected  Examined  Affected 
Total number  Mean (%)  Total number  Mean (%)  Total number  Mean (%) 
0 149 0 0 33 1 2.2 116 2 2
0.2 137 0 0 26 0 0 111 2 1.7
2 147 0 0 22 0 0 125 5 5.5
300 153 1 0.6 30 0 0 122 4 3.6
600 166 0 0 34 1 6.7 132 2 1.8

b) Group mean incidence for pups with extra ribs (Palmer et.al., 1975)

Dosage (mg/kg bw/day) Mean percent incidences of pups with extra lumber ribs*
0 18.6
0.2 33.1
2 21.3
300 12.9
600 15.3

* Young showing major malformations excluded

Executive summary:

A pre-natal developmental toxicity study is conducted with C10-13LAS, sodium salt in pregnant female CD rats. The test substance was administered to groups of 20 pregnant female rats via oral gavage at doses of 0, 0.2, 2, 300 and 600 mg/kg bw/day from gestation day 6 (GD 6) till GD 15. All animals were observed daily for clinical signs of toxicity and were weighed regularly throughout the gestation. On GD 17, animals were euthanised and uteri were examined for number of implantations, viable youngs and embryonic deaths (abortion or resorption sites). Ovaries were examined and number of corpora lutea were also recorded. Individual foetus was weighed and examined for external malformations. About one third of the foetuses were examined for visceral anomalies and remaining two thirds for skeletal anomalies. Moderate maternal toxicity was observed at the highest dose due to retarded weight gain and mortality. These effects were associated with the gastrointestinal disturbances. No instances of major, minor and skeletal malformations were observed up to the highest dose of 600 mg/kg bw/day with the exception of a marginal retardation of sternebral ossification at 600 mg/kg. Based on the results, the maternal toxicity and developmental toxicity NOAELs were determined to be 300 mg/kg bw/day based on retarded weight gain and mortality in treated females at higher dose and based on absence of major, minor and skeletal malformations at highest dose respectively (Palmer, 1975a).  

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The aim of this study was to evaluate the teratogenic potential of linear alkylbenzene sulphonate (LAS) in mice. Twenty female mice/dose were assigned into 5 groups and administered 0 (water), 0.2, 2.0, 300 and 600 mg/kg bw/day of test substance by oral gavage from day 6 to 15 of gestation. Throughout the experiment animals were observed daily for signs of malreaction and weighed regularly. After euthanasia on day 17, their uterine content was examined for number of implantations, viable young and embryonic deaths. Body weight, incidents of external malformation, visceral and skeletal anomalies were also recorded in pups. Non parametric methods were used for statistical analysis and on the basis of adverse effects, NOAEL value was derived for maternal toxicity and teratogenicity.
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
CD-1
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass., U.S.A. and St-Aubin-les-Elbeuf, France.
- Housing: Femlale mice (5 per cage) were housed in opaque plastic cages.
- Diet: Spratt’s Laboratory Diet No. 1, ad libitum.
- Drinking water: ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 1°C
- Humidity: 50 ± 5 % RH
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- Preparation of dosing solutions: Doses were prepared on daily basis as series of graded aqueous solutions for oral dosing.
- Administration of dosage: Standard volume of doses was administered orally by gastric intubation. Control animals were dosed with water.
- Duration of dosage: Doses were commenced on day 6 after detection of vaginal plug in mice and continued daily up to and including day 15 of gestation.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Mating was confirmed by the detection of vaginal plug in mice.
Duration of treatment / exposure:
Animals were dosed for 10 days [from Day 6 (after detection of vaginal plug) to Day 15].
Frequency of treatment:
daily from day 6 after detection of the vaginal plug to till day 15
Duration of test:
18 days
Dose / conc.:
0 mg/kg bw/day
Remarks:
(control)
Dose / conc.:
0.2 mg/kg bw/day
Dose / conc.:
2 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
600 mg/kg bw/day
No. of animals per sex per dose:
20 female mice per dose
Control animals:
yes
Details on study design:
- Dose selection rationale: Two doses formed the basis for safety evaluation (0.2 and 2.0 mg/kg bw/day) because the likely maximum human intake of detergent from ordinary kitchen use has been estimated at 0.14 mg/kg/day, thus providing factors of 1-2 times the human exposure level. Two further doses (300 and 600 mg/kg bw/day) were also investigated based on previous toxicity data suggesting that these would impair maternal economy and result in obvious adverse effects.
Maternal examinations:
All animals were observed daily for signs of malreaction and were weighed regularly throughout gestation. All animals that died, and survivors at termination, were dissected and examined for macroscopic changes
Ovaries and uterine content:
After euthanasia, uteri were dissected and examined to determine the number of:
a) Implants
b) Viable young
c) Embryonic deaths (abortion/resorption sites)
Fetal examinations:
After weighing, fetus were examined for external malformations, visceral anomalies by free and sectioning and skeletal examination was performed by alizarin staining.
Statistics:
Differences in mean values were statistically analyzed by non-parametric method i.e. Wilcoxon test.
Historical control data:
In CD-1 mice, percentage of major malformations, minor visceral anomalies and minor skeletal anomalies were recorded as 0.84%, 3.68% and 5.32%, respectively in 22389 examined fetuses.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Marked maternal toxicity was observed at 300 and 600 mg/kg bw/day. Toxic effects were generally associated with the gastrointestinal tract disturbances.
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
There was 90 and 35% mortality in 600 and 300 mg/kg bw/day does groups, respectively while no mortality was observed in 0.2 and 2 mg/kg bw/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain was retarded in the animals of 300 mg/kg bw/day and weight loss was observed at 600 mg/kg bw/day but due to high mortality rate at this dose, no conclusion can be drawn.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Marked maternal toxicity was observed in mice at 300 and 600 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
At 300 and 600 mg/kg bw/day, 4 and 1 litters were lost, respectively.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Post-implantation embryonic losses were 6.5%, 11.5%, 9.4%, 38.0% and 100% at 0, 0.2, 2, 300 and 600 mg/kg bw/day, respectively. No information on pre-implantation loss was provided in the research article.
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
Embryonic deaths were 0.8, 1.2, 1.2, 4.5 and 12.0 at 0, 0.2, 2, 300 and 600 mg/kg bw/day, respectively.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Other effects:
not specified
Details on maternal toxic effects:
Maternal toxic effects at 300 and 600 mg/kg bw/day were primarily associated with the gastrointestinal disturbances. Treatment at 300 and 600 mg/kg bw/day were associated with increased mortality of 35% and 90%, respectively in maternal animals along with marked decrease in mating index at 600 mg/kg bw/day. At 300 mg/kg bw/day weight gain was also retarded and weight loss was observed at 600 mg/kg bw/day but due to high mortality rate at this dose, no conclusion can be drawn.
Key result
Dose descriptor:
NOAEL
Effect level:
2 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: marked maternal toxicity at 300 and 600 mg/kg bw/day
Abnormalities:
not examined
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
Increased fetal loss was observed at maternally toxic doses (i.e. 300 and 600 mg/kg bw/day)

Changes in sex ratio:
not specified
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Reduced litter size was observed at maternally toxic doses (i.e. 300 and 600 mg/kg bw/day) due to total litter losses.
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Higher incidences of skeletal abnormalities were observed at 300 mg/kg bw/day.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Incidences of minor visceral anomalies were increased at 300 mg/kg bw/day.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
EMBRYO TOXICITY
Fetal weights: Although the fetal weight differed significantly at 0.2 mg/kg bw/day when compared to control group, these changes does not represent an adverse change because there was no consistent dose relationship and were invariably due to higher mean pub weight in the test group.

Fetal loss: Increased fetal loss was observed at maternally toxic doses (i.e. 300 and 600 mg/kg bw/day) due to total litter loses.

Litter size and weight: Reduced litter size was observed at maternally toxic doses (i.e. 300 and 600 mg/kg bw/day) due to total litter loses. This effect was a secondary consequence of maternal reaction as litter parameters were comparable with control values in mice. Litter weights were comparable across all doses when compared to the control group.

MALFORMATIONS:
No test substance related or statistically differences in the incidence of major malformations observed even a maternally toxic dosages but minor visceral anomalies were increased at 300 mg/kg bw/day (not significant statistically).

DEVELOPMENTAL VARIATIONS:
An increase in incidences of skeletal abnormalities were observed at 300 mg/kg bw/day with respect to both concurrent controls as well as laboratory standard range (2.2 – 30.5%).
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: higher incidence of skeletal and visceral anomalies at 600 mg/kg bw/day
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: skeletal and visceral anomalies
Description (incidence and severity):
higher incidence of skeletal and visceral anomalies were observed at 600 mg/kg bw/day
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
yes

Formula for calculation of mating index:

Mating index: (number of confirmed mating/ number of animals cohabitated) X 100

Table 1: Mating index of the maternal animals (Palmer et.al., 1975)

Dose Number of confirmed mating Number of animals cohabitated Mating index %
0 17 20 85
0.2 18 20 90
2 18 20 90
300 13 13 100
600 1 2 50

Table 2: Summary of adult performance (Palmer et.al., 1975)

Observations Number of animals at dosage (mg/kg bw/day)
0 0.2 2 300M 600M
Mated 20 20 20 20 20
Died 0 0 0 7 18
Non-pregnant 3 2 2 0 1
Total litter loss 0 0 0 4 1
With viable young 17 18 18 9 0
Body weight change - - - retarded gain loss

M: Marked maternal toxicity

Table 3: Group mean litter data (Palmer et.al., 1975)

Dosage (mg/kg bw/day) Number of litters Litter size viable young Embryogenic deaths Implantations Embryonic loss % Litter weight (g) Fetal weight (g)
Post-implantation
0 17 (A & B) 11.7 0.8 12.5 6.5 11.34 0.97
0.2 18 (A & B) 10.4 1.2 11.6 11.5 10.98 1.05a
2 18 (A & B) 11.4 1.2 12.7 9.4 11.6 1.02
300 13 (A) 7.9 4.5 12.4 38 - -
9 (B) 11.3 1.3 12.7 10.5 10.83 0.95
600 1 (A) 0 12 12 100 - -

A: Mean value includes all surviving animals showing evidence of implantation, including those with total litter loss

B: Mean value includes only animals bearing viable young at termination

a: Difference from controls statistically significant at Wilcoxon test (P < 0.05)

Table 4:

a) Group mean incidence for major and minor malformations (Palmer et.al., 1975)

Dosage (mg/kg bw/day) Number of young
Major malformations Minor malformations*
Gross or visceral Skeletal
Examined Affected Examined Affected Examined Affected
Total number Mean (%) Total number Mean (%) Total number Mean (%)
0 198 0 0 49 1 2 149 18 11.7
0.2 187 2 1 42 3 9.8 143 14 10.2
2 206 0 0 59 2 3.5 147 19 13.3
300 102 1 0.8 26 3 12.2 75 26 33.7
600 0 - - - - - - - -

b) Group mean incidence for pups with extra ribs (Palmer et.al., 1975)

Dosage (mg/kg/day) Mean percent incidence of pups with extra ribs*
Cervical Lumbar
0 32.4 19.1
0.2 41.3 13.3
2 17.3 21.2
300 40.8 6.7
600 - -

* Young showing major malformations excluded

Conclusions:
Administration of linear alkylbenzene sulphonate (LAS) to female CD-1 mice by oral gavage during days 6-15 of gestation at dose levels of 0, 0.2, 2, 300 and 600 mg/kg bw/day resulted in a NOAEL of 2 mg/kg bw/day for maternal toxicity (based on marked maternal toxicity at 300 and 600 mg/kg bw/day) in treated females as compare to controls. The NOAEL for teratogenicity was also established at 2 mg/kg bw/day (based on higher incidence of skeletal and visceral anomalies at 300 mg/kg bw/day).
Executive summary:

A pre-natal developmental toxicity study is conducted with C10-13LAS, sodium salt in pregnant female CD-1 mice. The test substance was administered to groups of 20 pregnant female mice via oral gavage at doses of 0, 0.2, 2, 300 and 600 mg/kg bw/day from gestation day 6 (GD 6) till GD 15. All animals were observed daily for clinical signs of toxicity and were weighed regularly throughout the gestation. On GD 17, animals were euthanised and uteri were examined for number of implantations, viable youngs and embryonic deaths (abortion or resorption sites). Ovaries were examined and number of corpora lutea were also recorded. Individual foetus was weighed and examined for external malformations. About one third of the foetuses were examined for visceral anomalies and remaining two thirds for skeletal anomalies. Marked maternal toxicity was observed in mice at 300 and 600 mg/kg bw/day. These effects were associated with the gastrointestinal disturbances. Total litter loss (abortion and / or total resorption) also occurred as secondary consequence of the primary effect on mother. Higher incidences of skeletal abnormalities were also detected in mice foetus at 300 mg/kg bw/day group. Because of the very wide range between the 2 mg/kg bw/day and 300 mg/kg bw/day doses, the maternal NOAEL of 2 mg/kg bw/day must be considered very conservative. At 300 mg/kg bw/day, the incidence of total litter loss was 20%; this was attributed to the high maternal toxicity observed at this dose. Among the nine animals with viable young, mean litter parameters, including litter size and foetal loss, and incidence of major malformations were not statistically different from controls. Minor anomalies, including gross or visceral and skeletal anomalies, were increased. At the 600 mg/kg bw/day, there were no live births. Based on these data, the developmental toxicity NOAEL was considered to be 300 mg/kg bw/day (Palmer, 1975b).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Karl THOMAE, Biberach an der Riss, Germany,
- Age at study initiation: Sexually mature
- Weight at study initiation: Approximately 200-300g mean weight approx. 223.7 g
- Fasting period before study: none
- Housing: Animals were singly housed in 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
- Relative humidity: 30-70%
- Photoperiod: 12 hours dark /12 hours light
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(doubly distilled)
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
- Amount of vehicle: 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.)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of test substance in the dosing solutions, homogeneity, and stability throughout the dosing period were all confirmed analytically. The homogeneity of the test substance was proven by visual inspection. The content of active ingredient was 99.4% before the beginning of the study. The reanalysis of the test substance proved its stability (content: 99.5%)
Details on mating procedure:
- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: Sperm in vaginal smear was referred to as day 0 of pregnancy
Duration of treatment / exposure:
Day 6 - 15 of gestation
Frequency of treatment:
Daily, once per day
Duration of test:
Up to day 21 of gestation
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
40 mg/kg bw/day
Dose / conc.:
120 mg/kg bw/day
Dose / conc.:
450 mg/kg bw/day
No. of animals per sex per dose:
40 dams per dosing group
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for route of administration: Oral route by gavage was selected because this route was considered the most closely equivalent to the typical route of human exposure.
- Dose selection rationale: Dose selection for this study were chosen based upon the results of a preliminary study designed to investigate the prenatal toxicity of MEA in Wistar rats following oral administration of MEA at dose levels of 0, 50, 150, 300, or 500 mg/kg bw/day.

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 approximately 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). On day 20 p.c., the first 25 animals/group were sacrificed in a randomized order and examined macroscopically. The fetuses were dissected from the uterus and further investigated with different methods. The other animals (15/group) were allowed to litter and rear their pups up to day 21 p.p. (post partum). On day 21 post partum (p.p.) or one of the following days the relevant dams and pups were sacrificed and examined macroscopically.
- Rationale for selection of species and strain: The rat is an acceptable model for developmental toxicity studies. The reported strain is susceptible to known developmental toxicants.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
All animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited. The nesting, littering, and lactation behavior of the dams with terminal sacrifice day 21 p.p. was generally evaluated in the mornings in connection with the daily clinical inspection of the dams. Only if there were any special findings (e.g., animal could not litter, umbilical cord not cut), these specific findings were documented with the dam concerned. The littering behavior of the relevant dams was also inspected on weekdays (except holidays) in the afternoons in addition to the evaluations in the mornings. These reevaluation were documented separately, but, as before, findings were only recorded with the dams concerned. Moreover, the duration of gestation, the number of live and dead pups at birth and litter size were recorded for the animals with terminal sacrifice on day 21 p.p.

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

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on Gestation day 0, 1, 3, 6, 8, 10, 13, 15, 17, and 20. Body weights of dams which littered were also determined on the day of parturition and on days 4, 7, 14, and 21 postpartum.

Corrected body weight gain (net maternal body weight change)
Furthermore, the corrected body weight gain was calculated for all animals with terminal sacrifice on Gestation 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: Yes
- Time schedule for examinations: Feed consumption was determined at 2-3 day intervals.
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.

Ovaries and uterine content:
Examinations of the dams at termination Dams with terminal sacrifice on day 20 p.c.
On GD day 20, 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)

Dams with terminal sacrifice on day 21 p.p.
On day 21 post partum (p. p.) the relevant dams were sacrificed by cervical dislocation. After the
dams had been sacrificed, the following examinations were carried out:
- gross - pathological examination
- staining of uterus according to Salewski for determination of the number of implantations
Fetal examinations:
EXAMINATION OF THE FETUSES:
Examination of the fetuses after dissection from the uterus
At necropsy each fetus was weighed, sexed and examined macroscopically for any external findings.
The sex was determined by observing the distance between the anus and the base of the genital tubercle and was later confirmed in all fetuses fixed in Bouin's solution by internal examination. If there were discrepancies between the "external" and the "internal" sex of a fetus, the fetus was finally sexed according to the appearance of its gonads. Furthermore, the viability of the fetuses and the condition
of the placentae, the umbilical cords, the fetal membranes and fluids were examined. Individual placental weights were recorded. After these examinations, approximately one half of the fetuses per dam was placed in ethyl alcohol and the other half was placed in Bouin's solution for fixation and further e valuation.

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

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

EVALUATION CRITERIA FOR ASSESSING SKELETONS AND ORGANS OF THE FETUSES
In the present investigations the following terms (definitions) were used for describing a change:
- Malformations (concerning external, soft tissue and skeletal observations)
Rare and/or probably lethal changes were classified as malformations (e.g. exencephaly, atresia ani, hernia umbilicalis).

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

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

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

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

PUP VIABILITY / MORTALITY
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods which will be described in detail before. The number and percentage of dead pups on the day of birth (day 0) and of pups dying between days 1-4, 5-7, 8-14 and 15-21 of the lactation period were determined; however, pups which died accidentally were not included in these calculations. The number of live pups/litter was calculated on the day of birth, and on lactation days 4, 7, 14 and 21.

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.

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

PUP CLINICAL OBSERVATIONS
The pups were examined each day for clinical symptoms (including gross-morphological findings).

PUP NECROPSY OBSERVATIONS
After sacrifice on day 21 p.p. or one of the following days (by means of CO2) or intercurrent death, the pups were examined externally, eviscerated and their organs were assessed macroscopically with special attention being paid to the urinary tract. After the macroscopic examination of the pups, the kidneys of each pup were placed into cassettes and fixed in 4% formaldehyde solution for a possible further examination by light microscopy. If there were notable findings or if abnormalities were found in the daily clinical observation of the animals after their delivery, the affected animals were, if it was deemed necessary, examined additionally using appropriate methods (e.g., skeletal staining according to modified Dawson's method and/or further processing of head according to Wilson's method. The stained skeletons were evaluated under a stereomicroscope or a magnifying glass. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation (with an exception of the kidneys (see above)).
Statistics:
Dunnett-Test was used for a simultaneous comparison of several dose groups with the control. The hypothesis of equal means was tested. This test was performed two-sided and was used for the
statistical evaluation of food consumption, body weights and body weight change (females and pups), corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, number of corpora lutea, number of implantations, number of resorptions and number of live fetuses; proportion of preimplantation loss, postimplantation loss, resorptions and live fetuses in each litter; litter mean fetal body weight and litter mean placental weight, duration of gestation and number of pups delivered per litter. For the body weight and the body weight change of the pups the mean weight of each litter was used for the statistical analysis (statistical unit = litter).

Fisher' s Exact Test was used for a pairwise comparison of each dose group with the control for the hypothesis of equal proportions. This test was performed one - sided was used for statistical evaluation of the following parameters: female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings, female fertility index, gestation index, females with liveborn, stillborn and with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy.

The Wilcoxon Test was used for a comparison of each dose group with the control for the hypothesis of equal medians. This test was performed one-sided and was used for the proportion of fetuses
with malformations, variations, retardations and/or unclassified observations in each litter and for the proportion of affected pups per litter with necropsy observations. If the results of these tests were significant, labels (*for ≤ 5 0.05, ** for p ≤ 0.01) were printed in the summary tables.
Indices:
The fertility and the gestation indices were calculated according to the following formula:

- Fertility index = (n pregnant animals/ n mated animals) x 100

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

Calculations of conception rate and pre- and post implantation 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

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

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

- 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
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
A single dam from the 40 mg MEA/kg/day group died during parturition and undelivered pups were found in the uterus. All other dams survived the test period
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reductions in mean body weight was observed in rats on GD 15, 17, and 20 at the highest dose level tested, 450 mg/kg bw/day. Body weight gains for these dams were also significantly reduced during GD 15 - 20 and 0-20.
Lactation body weights of the remaining 9-12 dams in all dose groups were slightly, but statistically decreased relative to controls; however, no adverse effect was observed on overall body weight gain during lactation at any dose level.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Dams administered 450 mg MEA/kg/day exhibited significant decreases in feed consumption on day 6-8 (8%) and 17-20 (5%). A statistically significant reduction in feed consumption (18%) was also noted for the remaining 450 mg/kg bw/day dams early in the lactation period on days 0-4.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
Maternal toxicity data:

Duration of Pregnancy:

The mean duration of gestation for the 21 postpartum (p.p.) females was 21.8 days, 21.6 days, 21.6 days, and 21.4 days for the control, the 40 mg/kg bw/day, 120 mg/kg bw/day and the 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/water 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, Description, severity, time of onset and duration of clinical signs: No signs that might be attributed to the test substance administered were detected during gestation and lactation periods. During gestation, piloerection was recorded for one high dose animal on day 13. Without any dose- response relationship insufficient nesting activity was observed for several dams of all groups. During lactation one dam of the 40 mg/kg 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 these findings are spontaneous in nature and cannot be attributed to the test substance administration.

Gross pathology incidence and severity:

There were no substance-related observations at necropsy in any of the dams. Hydrometra (a spontaneous finding) was recorded for one female of the control group, for 2 females of the 40 mg/kg/ day group, and 3 females of the 120 mg/kg bw/day group. These animals did not become pregnant. Edema of the lungs which has to be related to the termination of the rats was recorded for several dams of the control, low and intermediate groups without any relation to dosing. For the one low dose female that died intercurrently during parturition, undelivered pups were found in the uterus. Organ weight changes, particularly effects on total uterine weight: The uterus weights, which were determined for the animals with termination on day 20 of gestation only, were not influenced by the administration of the test substance. The differences between the groups is without biological relevance and do not show any dose-response relationship.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Details on maternal toxic effects:
Maternal toxic effects: yes

Details on maternal toxic effects:
Monoethanolamine caused some signs of maternal toxicity when administered by gavage to pregnant Wistar rats from days 6 - 15 of gestation at the highest dose level tested, 450 mg/kg bw/day. Maternal toxicity was substantiated by reduced food consumption, lower mean body weights and impaired body weight gain. The oral administration of monoethanolamine at 450 mg/kg bw/day or doses below this had no influence on resorption rate, number of live fetuses or pups/dam, mean fetal weight or pup body weights.
Key result
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: Based on statistically significant decreases in maternal body weights and feed consumption at 450 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant
Remarks on result:
other: No treatment-related effects were observed on reproductive parameters including pregnancy rate, number of corpora lutea, number of implantations, resorptions, litter size and number of dead fetuses
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Anascara was recorded in a single fetus administrating a dose of 450 mg/kg bw/day
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Asymmetrical (dumb-bell shaped or bipartite) thoracic vertebral bodies in few foetuses were observed. Minor skeletal variations relating to the thoracic vertebrae, ribs, and/or sternebrae were recorded at similar frequencies in all dose groups.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral malformations including microphthalmia, dilatation of the right or both heart ventricles, dextrocardia, unilobular lung, or hyper-/hypoplasia of the kidneys were noted sporadically throughout all dose groups, including the control and did not show any relation to dose level. Two visceral variations, dilated renal pelvis and hydroureter, occurred in several fetuses from all dose groups without any dose-response relationship.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
No signs of developmental toxicity occurred up to and including the highest dose level (450 mg/kg 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-related groups. All skeletal malformations, variations, or retardations which occurred did not show a clear dose-response relationship, can be found at comparable or even higher rates within the historical control and/or the differences between the groups are without biological significance.

Fetal data:
Litter size and weights

Of the females euthanized on day 21 p.p. the number of females with liveborn was 12, 10, 13, and 10 and the number of pups delivered was 165, 132, 170, and 125 for the control, the 40 mg/kg bw/day, 120 mg/kg bw/day and the 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 grams for all groups.

Number viable (number alive and number dead)

Dams with viable fetuses was 21, 20, 20 and 24 and the number of fetuses alive/dead were 293/0, 282/0, 263/0 and 311/0 for the control, the 40 mg/kg bw/day, 120 mg/kg bw/day and the 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 is also present at a low incidence in the historical control data. The external examination of the fetuses revealed no variations in any group. One unclassifed observation, fused placentae, was recorded in one fetus of the 40 mg/kg 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 group) and 75% (40 mg/kg bw/day group). 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/day group and 34 in the 450 mg/kg bw/day group. The number pregnant at caesarian-section was 21 in the control, 20 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, the 40 mg/kg bw/day, 120 mg/kg bw/day and the 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, the 40 mg/kg bw/day, 120 mg/kg bw/day and the 450 mg/kg bw/day groups, respectively. Mean late resorptions were 0.2, 0.3, 0.1 and 0.0 for the control, the 40 mg/kg bw/day, 120 mg/kg bw/day and the 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, the 40 mg/kg bw/day, 120 mg/kg bw/day and the 450 mg/kg bw/day groups, respectively. The mean number of implantation sites for the 21 p.p. females were 14.6, 14.6, 14.1 and 13.4 for the control, the 40 mg/kg bw/day, 120 mg/kg bw/day and the 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, the 40 mg/kg bw/day, 120 mg/kg bw/day and the 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, the 40 mg/kg bw/day, 120 mg/kg bw/day and the 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, the 40 mg/kg bw/day, 120 mg/kg bw/day and the 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 postimplantation losses. The females euthanized on day 21 p.p. showed no substance-associated effects on the postimplantation loss.

Number of corpora lutea
The mean numbers of corpora lutea were 16.1, 16.3, 15.6 and 15.7 for the control, the 40 mg/kg bw/day, 120 mg/kg bw/day and the 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, it seems very unlikely that the differences in pup body weight/body weight gain are substance-related.

Postnatal survival

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

Pup clinical observations
None of the pups of the different groups showed any clinical signs until termination. Pup necropsy observations: Only a few pups showed findings at necropsy. Post mortem autolysis, incisor sloped and dilated renal pelvis (1 high-dose pup) occurred in single pups of the control, the 40 and 450 mg/kg 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, can be found in a similar frequency in historical control data, and/or the differences between groups are without biological significance. In all groups signs of retardations (incomplete or missing ossification of vertebral bodies/arches and the sternebra(e)) were found. The differences between the groups, however, are not associated with the test substance administration. All of the skeletal retardations are to be found at a comparable frequency in the historical control data and most often a clear dose-response relationship is not present. The only statistically significant difference, an increased rate of total variations in the 120 mg/kg bw/day group, is without biological relevance because it shows no dose-dependence.
Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Remarks on result:
other: no treatment related adverse effect was observed on fetal survival, fetal/pup body weight, fetal sex ratio or incidence of external, skeletal and visceral malformations
Abnormalities:
effects observed, non-treatment-related
Localisation:
external: eye
skeletal: rib
skeletal: vertebra
visceral/soft tissue: urinary
visceral/soft tissue: cardiovascular
visceral/soft tissue: central nervous system
other: external: anasarca
Description (incidence and severity):
All the observed malformations were considered as non-treated related.
Key result
Developmental effects observed:
no

Table 1: Monoethanolamine pre, peri, postnatal toxicity: gestation bodyweight in rats (Hellwig and Liberacki, 1997)

Gestation day MEA (mg/kg bw/day)
0 40 120 450
  body weight (g) of damsa
0 228 ± 14 222 ± 14 226 ± 13 222 ± 12
6 258 ± 17 253 ± 16 256 ± 15 250 ± 14
10 275 ± 20 270 ± 18 272 ± 17 267 ± 16
15 310 ± 20 304 ± 21 305 ± 19 208 ± 19*
17 335 ± 22 329 ± 23 327 ± 22 321 ± 21*
20 385 ± 25 378 ± 28 373 ± 30 363 ± 25*
  Body weight gain (g)*
6-15 53 ± 6 51 ± 7 49 ± 8 48 ± 10
15-20 75 ± 10 73 ± 11 68 ± 15 65 ± 12*
0-20 157 ± 16 156 ± 19 147 ± 23 141 ± 20*
No. of dams: 33 30 33 34

a: Mean±SD.

* Statistically different from control (a = 0.05).

 

Table 2: Monoethanolamine pre, peri, postnatal toxicity: Lactation bodyweight in rats (Hellwig and Liberacki, 1997)

Gestation day MEA (mg/kg bw/day)
0 40 120 450
  body weight (g) of dams
0 303 ± 21a 281 ± 15* 283 ± 20* 275 ± 20*
4 320 ± 18 298 ± 21* 289 ± 21* 285 ± 17*
7 332 ± 21 309 ±27 300 ± 21* 297 ± 25*
14 327 ± 12 314 ±24 309 ± 18 309 ± 23
21 323 ± 17 304 ±22 304 ± 12* 302 ± 19*
  Body weight gain (g)
0-21 19 ± 18 23 ± 15 21 ± 15 27 ± 14
No. of dams: 12 9 13 10

a: Mean±SD.

* Statistically different from control (a = 0.05).

 

Table 3: Monoethanolamine pre, peri-, postnatal toxicity: reproductive parameters

 

Reproductive parameters MEA (mg/kg bw/day)
0 40 120 450
Cesarean section dams
No. deaths/No. Females 0/25 0/25 0/25 0/25
% Pregnant 84 80 80 %
No. of liners 21 20 20 24
Corpora lutea/dama 16 1 ± 1.42 16.3 ± 1.86 15.6 ± 2.54 15.7 ± 2.55
Implantations/dama 15.6 ± 1.50 15.3 ± 2.86 14.3 ± 3.80 13.8 + 3.67
Live fetuses/littera 14.0 ± 1.63 14.1 + 2.81 13.1 ± 3.47 13.0 ± 3.58
% Implantations resorteda 10.3 ± 7.22 7.3 ± 7.14 7.0 ± 8.92 6.3 ± 6.42
Fetal body weight (g)b 3.9 ± 0.2 3.9 ± 0.2 3.9 ± 0.3 3.9 ± 0.2
Placental weights (g)b 0.5 ± 0.04 0.5 ± 0.05 0.5 ± 0.06 0.5 ± 0.05
Fetal sex ratio (M:F, %) 54:46:00 51:49:00 56:44:00 49:51:00
Gravid uterine weight (g)a 81.2 ± 9.2 81.6 ± 15.2 76.0 ± 19.3 74.0 ± 18.5

a: Mean±SD.

b Mean of litter means (a = 0.05).

 

RESULTS  SUMMARY:

 

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

Test group 3 (450 mg/kgbw/day):

- statistically significantly reduced foodconsumption 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 post treatment days

15 - 20 p.c.

 

Test group 2 (120 mg/kgbw/day):

- no substance-related effectson dams, fetuses or pups

 

Test group 1 (40 mg/kgbw/day):

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

 

Thus,under the conditions of this study, Monoethanolamine pure caused some signs of maternal toxicity when administered by gavage to pregnant Wistar rats from days 6-15p.c.at the highest dose level tested (450mg/kgbw/day). Maternal toxicity was substantiated in this dose group by a reduced food consumption, lower mean body weights and impaired body weight gain.120 and 40mg/kgbw/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(450mg/kg bw/day).The reproductive parameters unaffected and neither the fetuses nor the pups showed an increased malformation rate or any indications for a substance-induced growth retardation; especially, the urinary tract of the rat fetuses/pups did not show any treatment-related findings.

 

Based on these study results,the no observable adverse effect level (NOAEL) on the maternal organism is 120 mg/kgbw/dayand 450 mg/kgbw/day for the progeny.

Conclusions:
Administration of monoethanolamine to female wistar rats by oral gavage during Days 6-15 of gestation at dose levels of 0, 40, 120 and 450 mg/kg bw/day resulted in a NOAEL of 450 mg/kg bw/day for developmental toxicity, based on no effects on reproductive parameters, incidences of visceral or skeletal malformations, or postnatal growth at any dose. The NOAEL for maternal toxicity was established at 120 mg/kg bw/day, based on statistically significant decreases in maternal body weights and feed consumption.
Executive summary:

A pre-natal developmental toxicity study was conducted with monoethanolamine (MEA) following methods comparable to OECD Guideline 414 in female Wistar rats. The test substance was administered to groups of 40 mated female rats by oral gavage once daily gestation day 6 (GD 6) till GD 15 at 0, 40, 120, and 450 mg/kg bw/day. All animals were observed daily for mortality and clinical observations. Body weights and feed consumption were determined regularly throughout the study. On GD 20, 25 dams/group were killed and necropsied, while the remaining 15 were allowed to litter and then killed on Day 21 of lactation. For the first 25 dams, the ovaries and uterine content (gravid uterus weight, number of corpora lutea, total implantations, and numbers of early and late resorptions, number and distribution of implantation sites) were examined. Individual foetuses were weighed, sexed, tagged and examined for external malformations and variations. All the foetuses in each lifter were examined for visceral alterations and about one-half of the foetuses were examined for skeletal defects. No test substance-related mortality was observed. Statistically significant reductions in mean body weight gain and feed consumption were observed in rats at the highest dose. A statistically significant reduction in feed consumption was observed in the high dose dams early in the lactation period. There were no significant differences in foetal sex ratio and mean placental or foetal weights in any of the doses and no treatment-related increases in the incidence of variations or malformations observed externally, viscerally, or at skeletal examination when compared to controls. Based on these results, a developmental toxicity NOAEL of 450 mg/kg bw/day was established in the rats based on absence of effects on the growth and development of the pups (Hellwig, 1997; BASF AG, 1994).  

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(Only 15 rabbits were used in the study, against the OECD guideline 414 recommendation of 20 animals.)
GLP compliance:
not specified
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hazelton Research Products, Inc. (Denver, PA), USA
- Age at study initiation: Not specified
- Weight at study initiation: Approximately 3.0-4 .0 kg
- Fasting period before study: none
- Housing: Wire-bottom cages
- Diet: AGWAY PROLAB Animal Diet Rabbit (Agway Inc., Waverly, NY); ad libitum
- Water: Municipal tap water; ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 20°C
- Relative humidity: 40-60%
- Photoperiod: 12 hours light/12 hours dark
Route of administration:
dermal
Vehicle:
water
Remarks:
(Milli-Q)
Details on exposure:
TEST SITE
- Area of exposure: Shaved skin of the back
- Type of wrap if used: Test substance was occluded with sterile gauze held in place by Lycra-Spandex jacket fitted with Velcro closures and lined with polyethylene film.

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

TEST MATERIAL
- Amount(s) applied: 2 mL/kg
- Constant volume or concentration used: Yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of monoethanolamine (MEA) in the dosing solutions (0.5-3.75%), stability throughout the dosing period, and homogeneity of the dosing solutions were confirmed analytically. No results of analytical verification are provided in the reference.
Details on mating procedure:
- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 2/1
- Length of cohabitation: Overnight
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: Copulation was referred to as day 0 of pregnancy
Duration of treatment / exposure:
Day 6 - 18 of gestation
Frequency of treatment:
6 hours/day, daily
Duration of test:
Up to day 29 of gestation
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
75 mg/kg bw/day
No. of animals per sex per dose:
15 dams/group
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 rabbits
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 3, 6, 9, 12, 15, 18, 24 and 29

FOOD CONSUMPTION: Yes
WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: Maternal liver and kidneys

OTHER: Hematologic determinations were done for all surviving rabbits prior to caesarian section and skin irritation was evaluated once daily during the post-dosing period.
Ovaries and uterine content:
The ovaries and uterine content were examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Uteri with no visible implantations were stained with a 10% sulfide solution.
Fetal examinations:
- External examinations and palate closure: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]
- Additional examinations: All fetuses were weighed and sex of all live fetuses was determined.

The heads of one half of the rabbit 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)
Statistics:
Continuous data were evaluated for homogeneity of variance using Levene's test. Based upon the outcome of this test, a parametric or nonparametric analysis of variance (ANOVA) was performed. If the ANOVA was significant, analysis by Dunnett's test, the Wilcoxen Rank-Sum test with Bonferroni's correction, or a pooled t test was performed as appropriate. The level of statistical significance was set a priori at a = 0.05. Nonparametric data were compared using Fischer's exact probability test.
Indices:
Pregnancy rate, implantations resorbed, litters with resorptions, fetal sex ratio were recorded. The details are provided in table 2 of “Any other information on results incl. tables” section.
Historical control data:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
effects observed, treatment-related
Description (incidence and severity):
A treatment-related severe skin irritation (erythema, edema, ecchymosis, necrosis, exfoliation, and crusting) was observed at the site of exposure in rabbits administered with 75 mg/kg bw/day of test substance. Subsequent to the dosing period, exfoliation, crusting, and areas of necrosis persisted. The skin of most of these rabbits began to heal as evidenced by scab formation late in the gestation period. Crusting, transient erythema, and edema were noted in a few rabbits administered 25 mg/kg bw/day. No significant dermal irritation or lesions were observed among rabbits administered 10 mg/kg bw/day.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Although no statistically identified changes were observed in either parameter, the average body weight gain of high-dose rabbits over the course of gestation was decreased when compared to that of the control and other dose groups, mainly due to weight loss or very little weight gain during the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
DERMAL IRRITATION: A treatment-related severe skin irritation (erythema, edema, ecchymosis, necrosis, exfoliation, and crusting) was observed at the site of exposure in rabbits administered with 75 mg/kg bw/day. Crusting, transient erythema, and edema were noted in a few rabbits administered 25 mg/kg bw/day.

BODY WEIGHT: The average body weight gain of rabbits administered with 75 mg/kg bw/day was consistently decreased (not statistically significant) due to weight loss or very little weight gain.

No adverse effect was observed in the feed consumption, hematologic parameters, liver or kidney weights at any dose level.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Details on maternal toxic effects:
Maternal toxic effects: yes

Details on maternal toxic effects:
Rabbits administered 75 mg/kg bw/day of MEA exhibited severe skin irritation (erythema, edema, ecchymosis, necrosis, exfoliation, and crusting) at the site of exposure. Subsequent to the dosing period, exfoliation, crusting, and areas of necrosis persisted. The skin of the most of these rabbits began to heal as evidenced by scab formation late in the gestation period. Crusting, transient erythema, and edema were noted in a few rabbits administered 25 mg/kg bw/day. No significant dermal irritation or lesions were observed among rabbits administered 10 mg/kg bw/day. There were also no significant treatment-related effects observed on feed consumption, hematologic parameters, or kidney and liver weights of MEA-exposed rabbits at any dose level tested. No statistically identified changes were observed in body weight and body weight gain, the average body weight gain of high-dose rabbits over the course of gestation was decreased when compared to that of the control and other dose groups, mainly due to weight loss or very little weight gain during the treatment period.

Details on maternal developmental toxicity
No females died, aborted, delivered early, or were removed from the test during the study.
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: dermal irritation – [local toxicity]
Remarks on result:
other: Based on dermal irritation at 25 and 75 mg/kg bw/day dose levels
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
haematology
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
other: No adverse effect was observed on body weight, food consumption, hematological parameters, organ weights and gross pathology
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant
Remarks on result:
other: No treatment-related effects were observed on reproductive parameters including pregnancy rate, number of corpora lutea, number of implantations, resorptions, litter size and number of dead fetuses
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Extra lumbar centrum and arches were observed in control animals; misaligned or fused thoracic centra, extra lumbar centrum and arches, and fused ribs were observed in 10 mg/kg bw/day dose group and diagonally displaced thoracic centra, missing thoracic arch, and a single missing rib were noted in 75 mg/kg bw/day dose group. No skeletal malformations were observed in 25 mg/kg bw/day dose group.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral malformations of ventricular septal defect, common opening at entry of the vessels of the heart, missing lung lobe were observed in controls. Dilated lateral cerebral ventricle with tissue depression, missing lung lobe were observed in all the tested dose levels (10, 25 and 75 mg/kg bw/day). Additionally, missing gallbladder was observed in 10 mg/kg bw/day dose group.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
No treatment-related effects were observed on reproductive parameters including 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 MEA exposed rabbits at any dose level when compared to controls.
There were no statistically or biologically significant treatment-related differences in the incidence of any fetal variation or malformation, or in the number of malformed fetuses in any dose group. Among control litters the following types of malformations were noted: ventricular septal defect (visceral malformation), common opening at entry of the vessels of the heart (visceral malformation), missing lung lobe (visceral malformation), missing gallbladder (visceral malformation), and extra lumbar centrum and arches (skeletal malformation).

Malformations observed in litters from rabbits given 10 mg/kg/day included visceral malformations of dilated lateral cerebral ventricle with tissue depression, missing lung lobe, missing gallbladder and skeletal malformations of misaligned or fused thoracic centra, extra lumbar centrum and arches, and fused ribs. Limited numbers of visceral malformations noted at 25 mg/kg/day included dilated lateral cerebral ventricle with tissue depression and missing lung lobe.

Malformations observed in fetuses from the 75 mg/kg bw/day dose group litters included the visceral malformations of dilated lateral cerebral ventricle with tissue depression, missing lung lobe and skeletal malformations of diagonally displaced thoracic centra, missing thoracic arch, and a single missing rib.
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Remarks on result:
other: no treatment related adverse effect was observed on fetal survival, fetal/pup body weight, fetal sex ratio or incidence of external, skeletal and visceral malformations
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: rib
skeletal: vertebra
visceral/soft tissue: gastrointestinal tract
visceral/soft tissue: central nervous system
visceral/soft tissue: respiratory system
Description (incidence and severity):
All the observed malformations were considered as non-treated related.
Key result
Developmental effects observed:
no

Table 1: Monoethanolamine (MEA) teratology: Maternal body weight measurements in rabbits (Liberacki et al., 1996)

Gestation day MEA (mg/kg/day)
0 10 25 75
Body weight (g) of dams
6 3884 ± 343a 3864 ± 396 3878 ± 403 3807 ± 477
  Body weight gain (g)
6-9 -25 ± 51 - 8 ± 110 -41 ± 42 -35 ± 58
9-12 15 ± 39 37 ± 36 24 ± 49 23 ± 33
12-15 24 ± 109 57 ± 40 49 ±49 -13 ± 83
15-18 - 3 ± 87 9 ± 59 10 ± 103 -15 ± 82
18-24 184 ± 91 176 ± 80 143 ± 50 142 ± 166
24-29 72 ± 140 43 ± 143 39 ± 116 3 ± 160
6-18 12 ± 214 95 ± 116 42 ± 137 -40 ± 162
0-29 399 ± 183 448 ± 207 356 ± 110 246 ± 409

a: Mean ± SD

Table 2: Monoethanolamine (MEA) teratology: Reproductive parameters in rabbits (Liberacki et al., 1996)

Reproductive parameters MEA (mg/kg bw/day)
0 10 25 75
Number deaths/number females 0/15 0/15 0/15 0/15
% pregnant 100 93 87 93
Number of litters 15 14 13 14
Corpora lutea/dama 10 ± 2 11 ± 3 10 ± 2 10 ± 2
Implantations/damb 9 ± 2 9 ± 4 9 ± 2 9 ± 2
Live fetuses/littera 8 ± 2 8 ± 4 8 ± 2 9 ± 2
% Implantations resorbed 2 (2/132) 4 (5/120) 2(2/115) 5 (7/131)
% litters with resorptions 13 (2/15) 29 (4/14) 15 (2/13) 21 (3/14)
Number dead fetuses/littera 0.3 ± 0.7 0.4 ± 0.6 0.3 ± 0.6 0.1 ± 0.5
Fetal body weight (g)b 43.4 ± 3.8 45.8 ± 7.7 43.4 ± 4.3 41.0 ± 6.6
Fetal sex ratio (M:F, %) 51:49:00 52:48:00 45:55:00 52:48:00
Gravid uterine weight (g)a 570 ± 95 545 ± 216 563 ± 93 575 ± 159

a: Mean + SD.

b: Mean of litter means ± SD.

Conclusions:
Administration of monoethanolamine (purity: 100%) to pregnant New Zealand White rabbits by dermal route for 6 hours per day during days 6-18 of gestation at dose levels of 0, 10, 25 and 75 mg/kg bw/day resulted in a NOAEL (maternal toxicity) of 75 mg/kg bw/day (systemic effects) and 10 mg/kg bw/day (local effects), based on skin irritation at higher two dose levels. The NOAEL (developmental toxicity, embryotoxicity and teratogenicity) was 75 mg/kg bw/day, based on no treatment related adverse effect on pregnancy rate, number of corpora lutea, number of implantations, resorptions, fetal survival, fetal/pup body weight, fetal sex ratio or incidence of external, skeletal and visceral malformations.
Executive summary:

A pre-natal developmental toxicity study was conducted with monoethanolamine (MEA) following methods comparable to OECD Guideline 414 in pregnant New Zealand White rabbits. The test substance was administered to groups of 15 pregnant rabbits via epidermal application for 6 h/day at doses of 0, 10, 25 and 75 mg/kg bw/day from gestation 6 (GD 6) till GD 18. On GD 21, dams were euthanized, and the foetuses were delivered by caesarean section. Weights of the maternal liver, kidneys, and gravid uteri were recorded. The number of corpora lutea, the numbers and position of implantations, resolutions, and live or dead foetuses were also noted. Uteri with no visible implantations were examined for evidence of early resorptions. No test substance-related mortality was observed. No treatment-related clinical or gross pathological observations were noted during the study at any dose, except for severe skin irritation at the mid and high doses at the site of application. No statistically significant effect was observed on body weights, feed consumption, liver or kidney weights at any dose level. There were also no significant differences in pregnancy rate, number of corpora lutea, number of implantations, resorptions, litter size, number of dead foetuses, foetal sex ratio, foetal body weight, or gravid uterine weight. No treatment-related increases in the incidence of variations or malformations observed externally, viscerally, or at skeletal examination when compared to controls. Based on these results, the developmental toxicity NOAEL was determined to be of 75 mg/kg bw/day based on absence of treatment related adverse effects on the growth and development of the pups (Liberacki, 1996a).  

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. (Kingston, NY), USA
- Age at study initiation: Not specified
- Weight at study initiation: Approximately 250-300 g
- Housing: Wire-bottom cages
- Diet: Certified Laboratory Rodent Chow (Ralston Purina Co., St. Louis, MO); ad libitum
- Water: Municipal tap water; ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C
- Relative Humidity: 40-60%
- Photoperiod: 12 hours light/12 hours dark
Route of administration:
dermal
Vehicle:
water
Remarks:
(Deionized)
Details on exposure:
TEST SITE
- Area of exposure: Shaved skin of the back
- Type of wrap if used: Test substance was occluded with 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 (if done): Water-dampened towel was used to wipe remaining test material off.
- Time after start of exposure: 6 hours


TEST MATERIAL
- Amount(s) applied: 1 mL/kg
- Constant volume or concentration used: Yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of monoethanolamine (MEA) in the dosing solutions (1-22.5%), stability throughout the dosing period, and homogeneity of the dosing solutions were confirmed analytically. No results of analytical verification are provided in the reference.
Details on mating procedure:
- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: Overnight
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: Sperm in vaginal smear was referred to as day 0 of pregnancy
Duration of treatment / exposure:
Day 6 - 15 of gestation
Frequency of treatment:
6 hours/day, daily
Duration of test:
Up to day 21 of gestation
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
75 mg/kg bw/day
Dose / conc.:
225 mg/kg bw/day
No. of animals per sex per dose:
30-45 rats/group
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.
The animals were exposed dermally to test substance for 6 hour per day at dose levels of 0, 10, 25, 75 and 225 mg/kg bw/day on day 6 to day 15 of gestation. The animals of the control group were treated in the same way with the vehicle (deionized distilled water).
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

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

FOOD CONSUMPTION: Yes

WATER CONSUMPTION: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: Maternal liver and kidneys

OTHER: The site of test substance application was assessed for indications of irritation following each daily treatment.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Uteri with no visible implantations were stained with a 10% sulfide solution.
Fetal examinations:
- External examinations and palate closure: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
- Additional examinations: All fetuses were weighed and 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. All fetuses were eviscerated and stained with alizarin red-S. Skeletal examinations were conducted only on the rat fetuses not selected for Bouin's examination.
Statistics:
Continuous data were evaluated for homogeneity of variance using Bartlett's test. 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, the Wilcoxen Rank-Sum test with Bonferroni's correction, or a pooled t test was performed as appropriate. The level of statistical significance was set a priori at a = 0.05. Nonparametric data was statistically treated using the Kruskal-Wallis test followed by the Mann-Whitney U test, 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.
Indices:
Pregnancy rate, implantations resorbed, litters with resorptions, fetal sex ratio were recorded. The details are provided in table 2 of “Any other information on results incl. tables” section.

Historical control data:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
effects observed, treatment-related
Description (incidence and severity):
A treatment-related increased incidence of skin irritation was observed at the site of exposure in rats administered with 225 mg/kg bw/day of test substance. However, no significant dermal irritation or lesions were observed among rats administered any other lower doses of test substance. There were no postmortem treatment-related effects observed in any dose group
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight gain of rats administered with 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 of test substance.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
DERMAL IRRITATION: A treatment-related increased incidence of skin irritation was observed at the site of exposure in rats administered with 225 mg/kg bw/day of test substance. However, no significant dermal irritation or lesions were observed among rats administered any other lower doses of test substance. There were no postmortem treatment-related effects observed in any dose group.

BODY WEIGHT: The body weight gain of rats administered with 225 mg/kg bw/day was significantly decreased during the exposure period.

No adverse effect was observed in the feed or water consumption, liver or kidney weights at any dose level. There were no postmortem treatment-related effects observed in any dose group.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Details on maternal toxic effects:
Maternal toxic effects: yes

Details on maternal toxic effects:
Rats administered with 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 of MEA. There were no postmortem treatment-related effects observed in any dose group. No significant differences were observed in the feed or water consumption of MEA 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 of MEA. No effects on liver or kidney weights were observed at any dose level.

Details on maternal developmental toxicity
There were no differences in pregnancy rate, number of corpora lutea, number of implantations, resorptions and number of dead fetuses.
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
other: dermal irritation – [local toxicity]
Remarks on result:
other: Based on dermal irritation and body weight changes at highest tested dose of 225 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Effect level:
225 mg/kg bw/day
Basis for effect level:
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant
Remarks on result:
other: No treatment-related effects were observed on pregnancy rate, number of corpora lutea, number of implantations, resorptions and number of dead fetuses in any of the tested dose levels
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
External malformation of microphthalmia was observed in control. A single fetus was malformed at 75 mg/kg bw/day dose group with multiple craniofacial malformations (external) consisting of micrognathia, cleft lip, soft palate and aglossia. No external malformations were observed in other dose groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
An extra cervical rib was observed in control and 25 mg/kg bw/day dose groups. No skeletal malformations were observed in other dose groups.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Retroesophageal right subclavian artery (visceral malformation) was observed in controls and 25 mg/kg bw/day dose group. No visceral malformations were observed in other dose groups.
Other effects:
not specified
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 MEA 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 (external malformation), retroesophageal right subclavian artery (visceral malformation), and an extra cervical rib (skeletal malformation). No malformed fetuses were observed in the 10 mg/kg bw/day dose group. Malformations observed in the 25 mg/kg bw/day dose group included retroesophageal right subclavian artery (visceral malformation) and an extra cervical rib (skeletal malformation). A single fetus was malformed in the 75 mg/kg bw/day dose group. This fetus had multiple craniofacial malformations (external 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.
Key result
Dose descriptor:
NOAEL
Effect level:
225 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Remarks on result:
other: no treatment related adverse effect was observed on fetal survival, fetal/pup body weight, fetal sex ratio or incidence of external, skeletal and visceral malformations
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
external: cranium
external: eye
external: face
skeletal: rib
visceral/soft tissue: cardiovascular
Description (incidence and severity):
All the observed malformations were considered as non-treated related.
Key result
Developmental effects observed:
no

Table 1: Monoethanolamine teratology: Maternal body weights in rats (Liberacki et al., 1996)

Gestation day MEA (mg/kg bw /day)
0 10 25 75 225
Body weight (g)
6 279 ± 16b 275 ± 15 276 ± 14 276 ± 10 280 ± 18
  Body weight gain (g)
6-9 9 ± 9 9 ± 11 7 ± 6 7 ± 6 5 ± 5
9-12 16 ± 6 16 ± 6 16 ± 7 16 ± 7 14 ± 8
12-16 29 ± 8 33 ± 7 30 ± 7 31 ± 10 25 ± 6
16-21 84+ 17 87 ± 13 85 ± 17 87 ± 23 82 ± 12
6-16 54 ± 14 58 ± 15 54 ± 10 54 ± 10 44 ± 10*
0-21 (total) 171 ± 27 178 ± 21 172 ± 28 173 ± 27 161 ± 22

b Mean + SD.

* Statistically different from control (α= 0.05)

 

Table 2: Monoethanolamine teratology: Reproductive parameters in rats (Liberacki et al.,1996)

Reproductive parameters MEA (mg/kg bw/day)
0 10 25 75 225
Number deaths/number females 0/45 0/30 0/30 0/30 0/30
% Pregnant 96 100 97 100 87
Number of litters 43 30 29 29a 26
Corpora lutea/damb 18 ± 3 19 ± 4 18 ± 3 19 ± 3 19 ± 3
Implantations/damb 16 ± 3 17 ± 2 15 ± 4 16 ± 2 16 ± 3
Live fetuses/litterb 15 ± 3 16 ± 2 15 ± 4 15 ± 3 15 ± 2
% Implantations resorbed 4 (29/666) 4 (21/496) 5 (23/448) 6 (26/462) 7 (29/408)
% Litters with resorptions 51 (22/43) 57 (17/30) 52 (15/29) 52 (15/29) 65 (17/26)
Number dead fetuses/litter 0 0 0 0 0
Fetal body weight (g)c 5.25 ± 0.41 5.16 ± 0.28 5.30 ± 0.35 5.17 ± 0.32 5.13 ± 0.25
Fetal sex ratio (M: F, %) 50:50:00 49:51:00 48:52:00 47:53:00 46:54:00
Gravid uterine weight (g) 107 ± 22 113 ± 13 106 ± 24 107 ± 19 103 ± 16

a: Decreased number of litters due to dam that delivered prior to scheduled necropsy.

b: Mean + SD.

c: Mean of litter means±SD.

Conclusions:
Administration of Monoethanolamine (purity: 100%) to pregnant Sprague-Dawley rats by dermal route for 6 hours per day during days 6-15 of gestation at dose levels of 0, 10, 25, 75 and 225 mg/kg bw/day resulted in a NOAEL (maternal toxicity) of 75 mg/kg bw/day, based on significant decrease in body weight and skin irritation and NOAEL (developmental toxicity, embryotoxicity and teratogenicity) of 225 mg/kg bw/day, based on no treatment related adverse effect on pregnancy rate, number of corpora lutea, number of implantations, resorptions, fetal survival, fetal/pup body weight, fetal sex ratio or incidence of external, skeletal and visceral malformations.
Executive summary:

A pre-natal developmental toxicity study was conducted with monoethanolamine (MEA) following methods comparable to OECD Guideline 414 in pregnant Sprague-Dawley rats. The test substance was administered to groups of 30-35 pregnant rats via epidermal application for 6 h/day at doses of 0, 10, 25, 75 and 225 mg/kg bw/day from gestation 6 (GD 6) till GD 15. On GD , dams were euthanized, and the foetuses were delivered by caesarean section. Weights of the maternal liver, kidneys, and gravid uteri were recorded. The number of corpora lutea, the numbers and position of implantations, resolutions, and live or dead foetuses were also noted. Uteri with no visible implantations were examined for evidence of early resorptions. No test substance-related mortality was observed. No treatment-related clinical or gross pathological observations were noted during the study at any dose, except for severe skin irritation at the mid and high doses at the site of application. No statistically significant effect was observed on body weights, feed consumption, liver or kidney weights at any dose level. There were also no significant differences in pregnancy rate, number of corpora lutea, number of implantations, resorptions, litter size, number of dead foetuses, foetal sex ratio, foetal body weight, or gravid uterine weight. No treatment-related increases in the incidence of variations or malformations observed externally, viscerally, or at skeletal examination when compared to controls. Based on these results, the developmental toxicity NOAEL was determined to be of 225 mg/kg bw/day based on absence of treatment related adverse effects on the growth and development of the pups (Liberacki, 1996b).  

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The aim of this study was to evaluate the teratogenic potential of linear alkylbenzene sulphonate (LAS) in rabbits. Thirteen female rabbits/dose were divided into 5 groups and administered 0 (water), 0.2, 2.0, 300 and 600 mg/kg bw/day of test substance by oral gavage from day 6 to 18 of gestation. Throughout the experiment animals were observed daily for signs of malreaction and weighed regularly. After euthanasia on day 29, their uterine content was examined for number of implantations, viable young and embryonic deaths. Ovaries of the rabbits were also examined and number of corpora lutea were counted. Body weight, incidents of external malformation, visceral and skeletal anomalies were also recorded in pups. Non parametric methods were used for statistical analysis and on the basis of adverse effects, NOAEL value was derived for maternal toxicity and teratogenicity.
GLP compliance:
not specified
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: East Anglian Rabbitries, Colchester, England
- Housing: Rabbits were housed individually in the metal cages equipped with wire mesh floor.
- Diet: Rabbits SG-1 diet, ad libitum.
- Drinking water: ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 2°C
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- Preparation of dosing solutions: Doses were prepared on daily basis as series of graded aqueous solutions for oral dosing.
- Administration of dosage: Standard volume of doses was administered orally by gastric intubation. Control animals were dosed with water.
- Duration of dosage: Doses were commenced on day 6 after observation of coitus in rabbits and continued daily up to and including day 18 of gestation.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Mating was confirmed by observation of coitus in rabbits.
Duration of treatment / exposure:
Animals were dosed for 13 days [from Day 6 (after observation of coitus) to Day 18].

Frequency of treatment:
daily from day 6 after observation of coitus to till day 18
Duration of test:
30 days
Dose / conc.:
0 mg/kg bw/day
Remarks:
(control)
Dose / conc.:
0.2 mg/kg bw/day
Dose / conc.:
2 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
600 mg/kg bw/day
No. of animals per sex per dose:
13 female rabbits per dose
Control animals:
yes
Details on study design:
- Dose selection rationale: Two doses formed the basis for safety evaluation (0.2 and 2.0 mg/kg bw/day) because the likely maximum human intake of detergent from ordinary kitchen use has been estimated at 0.14 mg/kg/day, thus providing factors of 1-2 times the human exposure level. Two further doses (300 and 600 mg/kg bw/day) were also investigated based on previous toxicity data suggesting that these would impair maternal economy and result in obvious adverse effects.
Maternal examinations:
All animals were observed daily for signs of malreaction and were weighed regularly throughout gestation. All animals that died, and survivors at termination, were dissected and examined for macroscopic changes.
Ovaries and uterine content:
After euthanasia, uteri were dissected and examined to determine the number of:
a) Implants
b) Viable young
c) Embryonic deaths (abortion/resorption sites)

Ovaries were examined and number of corpora lutea were also recorded.
Fetal examinations:
After weighing, fetuses were examined for external malformations followed by their dissection and examined for internal organ abnormalities and gonadal inspection for sex determination. The carcasses were preserved in alcohol for subsequent clearing, alizarin staining and skeletal examination.
Statistics:
Differences in mean values were statistically analyzed by non-parametric method i.e. Wilcoxon test.
Historical control data:
In rabbits, percentage of major malformations, minor visceral anomalies and minor skeletal anomalies were recorded as 0.74%, 2.53% and 8.60%, respectively in 36508 examined fetuses.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Marked maternal toxicity was observed at 300 and 600 mg/kg bw/day. Toxic effects were generally associated with the gastrointestinal tract disturbances and affected rabbits showed diarrhea, anorexia, weight loss and cachexia prior to death.
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
There was 100, 84.6% and 7.6% mortality observed at 600, 300 and 2 mg/kg bw/day, respectively while no mortality was observed at 0.2 mg/kg bw/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight loss was observed at 300 and 600 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
At 0.2, 2 and 300 mg/kg bw/day, 1, 1 and 2 litters were lost, respectively.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
Higher instance of embryonic death (8.5) was observed at 300 mg/kg bw/day.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Other effects:
not specified
Details on maternal toxic effects:
Marked maternal toxic effects were observed at 300 and 600 mg/kg bw/day which were primarily associated with the gastrointestinal disturbances and affected rabbits showed diarrhea, anorexia, weight loss and cachexia prior to death. Maternal mortality rate was 100, 84.6% and 7.6% in 600, 300 and 2 mg/kg bw/day doses groups, respectively while no mortality was observed at 0.2 mg/kg bw/day. Higher instance of embryogenic death was (8.5) was observed at 300 mg/kg bw/day. No effect was observed on the mating index.

Pre-implantation embryonic losses were 12.6%, 17.7%, 17.0%, and 12.5%at 0, 0.2, 2 and 300 mg/kg bw/day, respectively.

Post-implantation embryonic losses were 14.2%, 15.4%, 20.0% and 100% at 0, 0.2, 2 and 300 mg/kg bw/day, respectively.

Embryonic deaths were 1.7, 1.0, 1.4, and 8.5 at 0, 0.2, 2 and 300 mg/kg bw/day, respectively.
Key result
Dose descriptor:
NOAEL
Effect level:
2 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: marked maternal toxicity and mortality at 300 and 600 mg/kg bw/day
Abnormalities:
not examined
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
Increased fetal loss was observed at maternally toxic doses (i.e. 300 and 600 mg/kg bw/day)
Changes in sex ratio:
not specified
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Reduced litter size was observed at maternally toxic doses (i.e. 300 and 600 mg/kg bw/day) due to total litter losses.
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
EMBRYO TOXICITY
Fetal weights: No test substance related / statistically significant differences were observed in treated groups as compared to control group.

Fetal loss: Increased fetal loss was observed at maternally toxic doses (i.e. 300 and 600 mg/kg bw/day) due to total litter losses and number of young pups available for examination and adequate assessment of results were insufficient at these doses.

Litter size and weight: Reduced litter size was observed at maternally toxic doses (i.e. 300 and 600 mg/kg bw/day) due to total litter losses. Litter weights were comparable across all doses when compared to the control group.

MALFORMATIONS:
At maternally toxic dosages, number of young pups available for examination / adequate assessment of results were insufficient. No test substance related / statistically significant differences in the incidence of major malformations and minor anomalies were observed at 0.2 and 2 mg/ kg bw/day groups on comparison to the control group.

DEVELOPMENTAL VARIATIONS:
At maternally toxic dosages, number of young pups available for examination / adequate assessment of results were insufficient. No test substance related / statistically significant differences in the incidence of skeletal abnormalities were observed at 0.2 and 2 mg/ kg bw/day on comparison to the control group.
Key result
Dose descriptor:
NOAEL
Effect level:
2 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: no test substance related differences in the incidence of skeletal abnormalities, major malformations and minor anomalies were observed at this dose. Increased fetal mortality was observed at the two higher doses due to maternal toxicity.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Formula for calculation of mating index:

Mating index: (number of confirmed mating/ number of animals cohabitated) X 100

Table 1: Mating index of the maternal animals (Palmer et.al., 1975)

Dose Number of confirmed mating Number of animals cohabitated Mating index %
0 10 11 90
0.2 13 13 100
2 12 12 100
300 2 2 100
600# - - -

 #: Mating index cannot be calculated as all animals died at this dose.

Table 2: Summary of adult performance (Palmer et.al., 1975)

Observations Number of animals at dosage (mg/kg bw/day)
0 0.2 2 300M 600M
Mated 13 13 13 13 13
Died 2a 0 1 11 13
Non-pregnant 1 0 0 0 0
Total litter loss 1b 1 1 2 0
With viable young 9 12 11 0 0
Body weight change - - - Loss loss

a: Includes one animal dying of intubation error

b: Excluded from subsequent calculations due to pus in thoracic cavity and uterus

M: Marked maternal toxicity

Table 3: Group mean litter data (Palmer et.al., 1975)

Dosage (mg/kg bw/day) Number of litters  Litter size viable young  Embryogenic deaths  Implantations  Corpora lutea  Embryonic loss %  Litter weight (g) Fetal weight (g)
Pre-implantation  Post-implantation 
0 9 (A & B) 8.3 1.7 10 11.3 12.6 14.2 321.7 39.3
0.2 13 (A) 7.8 1 8.8 10.6 17.7 15.4 - -
12 (B) 8.5 0.8 9.3 10.7 13.4 8.4 323.3 38.5
2 12 (A) 7.3 1.4 8.8 11 17 20 - -
11 (B) 8 1.3 9.3 10.5 11.9 12.8 314.8 40.1
300 2 (A) 0 8.5 8.5 10 12.5 100 - -
600 No survivors - - - - - - - -

A: Mean value includes all surviving animals showing evidence of implantation, including those with total litter loss

B: Mean value includes only animals bearing viable young at termination

Table 4:

a)  Group mean incidence for major and minor malformations (Palmer et.al., 1975)

Dosage (mg/kg bw/day) Number of young
Major malformations Minor malformations*
Gross or visceral Skeletal
Examined Affected Examined Affected Examined Affected
Total number Mean (%) Total number Mean (%) Total number Mean (%)
0 75 3 3 72 7 11 72 8 11.9
0.2 102 0 0 102 3 3.3 102 7 8
2 88 1 1.3 87 1 1 87 8 9.5
300 0 - - - - - - - -
600 0 - - - - - - - -

b)  Group mean incidence for pups with extra ribs (Palmer et.al., 1975)

Dosage (mg/kg/day) Mean percent incidence of pups with extra ribs*
Lumbar
0 61
0.2 56.1
2 72.8
300 -
600 -

* Young showing major malformations excluded

Conclusions:
Administration of linear alkylbenzene sulphonate (LAS) to female NZW rabbits by oral gavage during days 6-18 of gestation at dose levels of 0, 0.2, 2, 300 and 600 mg/kg bw/day resulted in a NOAEL of 2 mg/kg bw/day for maternal toxicity (based on marked maternal toxicity and mortality at higher dose levels of 300 and 600 mg/kg bw/day) in treated females as compared to controls. The NOAEL for teratogenicity was also established at 2 mg/kg bw/day (based on no test substance related / statistically significant differences in the incidence of skeletal abnormalities, major malformations and minor anomalies at this dose).
Executive summary:

A pre-natal developmental toxicity study is conducted with C10-13LAS, sodium salt in pregnant New Zealand White rabbits. The test substance was administered to groups of 13 pregnant female rabbits via oral gavage at doses of 0, 0.2, 2, 300 and 600 mg/kg bw/day from gestation day 6 (GD 6) till GD 18. All animals were observed daily for clinical signs of toxicity and were weighed regularly throughout the gestation. After euthanasia on day 29, their uterine content was examined for number of implantations, viable young and embryonic deaths. Ovaries of the rabbits were also examined and number of corpora lutea were counted. Body weight, incidents of external malformation, visceral and skeletal anomalies were also recorded in pups. Marked maternal toxicity was observed at 300 and 600 mg/kg bw/day which were primarily associated with the gastrointestinal disturbances and affected rabbits showed diarrhoea, anorexia, weight loss and cachexia prior to death. Maternal mortality rate was 100, 84.6% and 7.6% at 600, 300 and 2 mg/kg bw/day, respectively while no mortality was observed at 0.2 mg/kg bw/day. Higher instance of embryogenic death was (8.5) was observed at 300 mg/kg bw/day. No effect was observed on the mating index. Increased foetal loss was observed at maternally toxic doses (300 and 600 mg/kg bw/day) due to total litter losses and number of young pups available for examination and adequate assessment of growth anomalies or malformations were insufficient at these doses. No test substance related / statistically significant differences in the incidence of major malformations and minor anomalies were observed at 0.2 and 2 mg/ kg bw/day in comparison to the control group.

Based on the results, the maternal toxicity and developmental toxicity NOAELs were determined to be 2 mg/kg bw/day (Palmer, 1975c).  

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
chronic
Species:
mouse
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
chronic
Species:
rabbit
Additional information

Developmental toxicity

Study 1 (C10-13 LAS):

 

A pre-natal developmental toxicity study is conducted with C10-13LAS, sodium salt in pregnant female CD rats. The test substance was administered to groups of 20 pregnant female rats via oral gavage at doses of 0, 0.2, 2, 300 and 600 mg/kg bw/day from gestation day 6 (GD 6) till GD 15. All animals were observed daily for clinical signs of toxicity and were weighed regularly throughout the gestation. On GD 17, animals were euthanised and uteri were examined for number of implantations, viable youngs and embryonic deaths (abortion or resorption sites). Ovaries were examined and number of corpora lutea were also recorded. Individual foetus was weighed and examined for external malformations. About one third of the foetuses were examined for visceral anomalies and remaining two thirds for skeletal anomalies. Moderate maternal toxicity was observed at the highest dose due to retarded weight gain and mortality. These effects were associated with the gastrointestinal disturbances. No instances of major, minor and skeletal malformations were observed up to the highest dose of 600 mg/kg bw/day. Based on the results, the maternal toxicity and developmental toxicity NOAELs were determined to be 300 mg/kg bw/day (based on retarded weight gain and mortality in treated females at higher dose) and 600 mg/kg bw/day (based on absence of major, minor and skeletal malformations at highest dose) respectively (Palmer, 1975a).  

Study 2 (C10-13 LAS):

 

A pre-natal developmental toxicity study is conducted with C10-13LAS, sodium salt in pregnant female CD-1 mice. The test substance was administered to groups of 20 pregnant female mice via oral gavage at doses of 0, 0.2, 2, 300 and 600 mg/kg bw/day from gestation day 6 (GD 6) till GD 15. All animals were observed daily for clinical signs of toxicity and were weighed regularly throughout the gestation. On GD 17, animals were euthanised and uteri were examined for number of implantations, viable youngs and embryonic deaths (abortion or resorption sites). Ovaries were examined and number of corpora lutea were also recorded. Individual foetus was weighed and examined for external malformations. About one third of the foetuses were examined for visceral anomalies and remaining two thirds for skeletal anomalies. Marked maternal toxicity was observed in mice at 300 and 600 mg/kg bw/day. These effects were associated with the gastrointestinal disturbances. Total litter loss (abortion and / or total resorption) also occurred as secondary consequence of the primary effect on mother. Higher incidences of skeletal abnormalities were also detected in mice foetus at 300 mg/kg bw/day group. Because of the very wide range between the 2 mg/kg bw/day and 300 mg/kg bw/day doses, the maternal NOAEL of 2 mg/kg bw/day must be considered very conservative. At 300 mg/kg bw/day, the incidence of total litter loss was 20%; this was attributed to the high maternal toxicity observed at this dose. Among the nine animals with viable young, mean litter parameters, including litter size and foetal loss, and incidence of major malformations were not statistically different from controls. Minor anomalies, including gross or visceral and skeletal anomalies, were increased. At the 600 mg/kg bw/day, there were no live births. Based on these data, the developmental toxicity NOAEL was considered to be 300 mg/kg bw/day (Palmer, 1975b).

Study 3 (MEA):

 

A pre-natal developmental toxicity study was conducted with monoethanolamine (MEA) following methods comparable to OECD Guideline 414 in female Wistar rats. The test substance was administered to groups of 40 mated female rats by oral gavage once daily gestation day 6 (GD 6) till GD 15 at 0, 40, 120, and 450 mg/kg bw/day. All animals were observed daily for mortality and clinical observations. Body weights and feed consumption were determined regularly throughout the study. On GD 20, 25 dams/group were killed and necropsied, while the remaining 15 were allowed to litter and then killed on Day 21 of lactation. For first 25 dams, the ovaries and uterine content (gravid uterus weight, number of corpora lutea, total implantations, and numbers of early and late resorptions, number and distribution of implantation sites) were examined. Individual foetuses were weighed, sexed, tagged and examined for external malformations and variations. All the foetuses in each lifter were examined for visceral alterations and about one-half of the foetuses were examined for skeletal defects. No test substance-related mortality was observed. Statistically significant reductions in mean body weight gain and feed consumption were observed in rats at the highest dose. A statistically significant reduction in feed consumption was observed in the high dose dams early in the lactation period. There were no significant differences in foetal sex ratio and mean placental or foetal weights in any of the doses and no treatment-related increases in the incidence of variations or malformations observed externally, viscerally, or at skeletal examination when compared to controls. Based on these results, a developmental toxicity NOAEL of 450 mg/kg bw/day was established in the rats based on absence of effects on the growth and development of the pups (Hellwig, 1997; BASF AG, 1994).

Study 4 (MEA):

 

A pre-natal developmental toxicity study was conducted with monoethanolamine (MEA) following methods comparable to OECD Guideline 414 in pregnant New Zealand White rabbits. The test substance was administered to groups of 15 pregnant rabbits via epidermal application for 6 h/day at doses of 0, 10, 25 and 75 mg/kg bw/day from gestation 6 (GD 6) till GD 18. On GD 21, dams were euthanized, and the foetuses were delivered by caesarean section. Weights of the maternal liver, kidneys, and gravid uteri were recorded. The number of corpora lutea, the numbers and position of implantations, resolutions, and live or dead foetuses were also noted. Uteri with no visible implantations were examined for evidence of early resorptions. No test substance-related mortality was observed. No treatment-related clinical or gross pathological observations were noted during the study at any dose, except for severe skin irritation at the mid and high doses at the site of application. No statistically significant effect was observed on body weights, feed consumption, liver or kidney weights at any dose level. There were also no significant differences in pregnancy rate, number of corpora lutea, number of implantations, resorptions, litter size, number of dead foetuses, foetal sex ratio, foetal body weight, or gravid uterine weight. No treatment-related increases in the incidence of variations or malformations observed externally, viscerally, or at skeletal examination when compared to controls. Based on these results, the developmental toxicity NOAEL was determined to be of 75 mg/kg bw/day based on absence of treatment related adverse effects on the growth and development of the pups (Liberacki, 1996a).

Study 5 (MEA):

A pre-natal developmental toxicity study was conducted with monoethanolamine (MEA) following methods comparable to OECD Guideline 414 in pregnant Sprague-Dawley rats. The test substance was administered to groups of 30-35 pregnant rats via epidermal application for 6 h/day at doses of 0, 10, 25, 75 and 225 mg/kg bw/day from gestation 6 (GD 6) till GD 15. On GD , dams were euthanized, and the foetuses were delivered by caesarean section. Weights of the maternal liver, kidneys, and gravid uteri were recorded. The number of corpora lutea, the numbers and position of implantations, resolutions, and live or dead foetuses were also noted. Uteri with no visible implantations were examined for evidence of early resorptions. No test substance-related mortality was observed. No treatment-related clinical or gross pathological observations were noted during the study at any dose, except for severe skin irritation at the mid and high doses at the site of application. No statistically significant effect was observed on body weights, feed consumption, liver or kidney weights at any dose level. There were also no significant differences in pregnancy rate, number of corpora lutea, number of implantations, resorptions, litter size, number of dead foetuses, foetal sex ratio, foetal body weight, or gravid uterine weight. No treatment-related increases in the incidence of variations or malformations observed externally, viscerally, or at skeletal examination when compared to controls. Based on these results, the developmental toxicity NOAEL was determined to be of 225 mg/kg bw/day based on absence of treatment related adverse effects on the growth and development of the pups (Liberacki, 1996b).

Study 6 (C10 -13 LAS):

 

A pre-natal developmental toxicity study is conducted with C10-13LAS, sodium salt in pregnant New Zealand White rabbits. The test substance was administered to groups of 13 pregnant female rabbits via oral gavage at doses of 0, 0.2, 2, 300 and 600 mg/kg bw/day from gestation day 6 (GD 6) till GD 18. All animals were observed daily for clinical signs of toxicity and were weighed regularly throughout the gestation. After euthanasia on day 29, their uterine content was examined for number of implantations, viable young and embryonic deaths. Ovaries of the rabbits were also examined and number of corpora lutea were counted. Body weight, incidents of external malformation, visceral and skeletal anomalies were also recorded in pups. Marked maternal toxicity was observed at 300 and 600 mg/kg bw/day which were primarily associated with the gastrointestinal disturbances and affected rabbits showed diarrhoea, anorexia, weight loss and cachexia prior to death. Maternal mortality rate was 100, 84.6% and 7.6% at 600, 300 and 2 mg/kg bw/day, respectively while no mortality was observed at 0.2 mg/kg bw/day. Higher instance of embryogenic death was (8.5) was observed at 300 mg/kg bw/day. No effect was observed on the mating index. Increased foetal loss was observed at maternally toxic doses (300 and 600 mg/kg bw/day) due to total litter losses and number of young pups available for examination and adequate assessment of growth anomalies or malformations were insufficient at these doses. No test substance related / statistically significant differences in the incidence of major malformations and minor anomalies were observed at 0.2 and 2 mg/ kg bw/day in comparison to the control group. Based on the results, the maternal toxicity and developmental toxicity NOAELs were determined to be 2 mg/kg bw/day (Palmer, 1975c).

Justification for classification or non-classification

Based on the reproductive and developmental toxicity studies with the dissociation products of MEA-LAS (MEAand LAS) in rats, mice and rabbits, no classification is warranted for this endpoint according to EU CLP (1272/2008/EC) criteria.