Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to reproduction: NOAEL reproduction (m, f): 1000 mg/kg bw (OECD 421, GLP)

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 May - 08 Sep 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Arbeit, Gesundheit und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 289-339 g (males) and 170-200 g (females)
- Housing: animals were housed in groups of up to three in open macrolon cages type 2000P (TechniPlast).
- Diet: maintenance diet for rats and mice, No. 1324 TPF, ad libitum. Dams received breeding diet for rats and mice, No. 1314 TPF, ad libitum
- Water: sterilized community tap water, ad libitum
- Acclimation period: 14-17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: 1% sodium carboxymethyl cellulose + 0,1% Polysorbate 80 (Tween 80), diluted in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test item and vehicle preparations were conducted weekly; the test item solution was prepared fresh on the day of application.

VEHICLE
- Justification for use and choice of vehicle (if other than water): As the test item’s solubility in water is poor, corn oil was used as an organic solvent for the preceding dose range finding study. Although the solubility of the test item in corn oil was sufficient, its oral application resulted in severe respiratory effects (laboured breathing, wheezing breath sounds) in all rats of the high dose group (1000 mg/kg body mass). The study director and the study monitor concluded that the physical (high surface activity) rather than the chemical properties of the test material when applied orally in this formulation lead to the effects observed in study. Based on an assessment of the physiochemical properties of the test material an adapted formulation from one of the acute toxicological studies was considered safe for application. The test item was applied as an emulsion in a watery solution at the three dosages specified in the study plan.
- Amount of vehicle (if gavage): 4 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until indications for mating were detected
- Proof of pregnancy: vaginal plug / sperm in vaginal referred to as Day 0 of pregnancy
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
(P) Males: 2 weeks before mating and continued throughout the mating period until the study was terminated
(P) Females: 2 weeks before mating, the variable time to conception, the duration of pregnancy and at least 4 days after delivery, up to and including the day before scheduled termination of the in-life phase. Therefore the duration of the study following acclimatisation depended on the female performance: 14 days pre-mating, up to 14 days until mating, an average of 21 days of gestation, and a minimum of 4 days of lactation.
Frequency of treatment:
daily, 7 days/week
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previously performed dose range finding study (OECD 407), the test item was administered in corn oil at three dosages up to 1000 mg/kg body mass over a time period of at least 40 days and produced irritating but no observable toxic effects in the test animals. Although its oral application resulted in severe respiratory effects (laboured breathing, wheezing breath sounds) in all rats of the high dose group (1000 mg/kg body mass), it was concluded that the physical (high surface activity) rather than the chemical properties of the test material lead to the effects observed in study. Therefore, the vehicle formulation was changed as described and 1000 mg/kg was determined as high dose for this study.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly and once before beginning of application

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly and once before beginning of application. Females during pregnancy: Day 0, 7, 14, 20, within 24h post parturition and Day 4 post partum.

FOOD CONSUMPTION: Yes
- Time schedule: at least once weekly

WATER CONSUMPTION: Yes
- Time schedule: twice weekly
Sperm parameters (parental animals):
Parameters examined in male parental animals: testis weight, epididymis weights. In high dose and control animals additionally performed: histology with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in offspring: number and sex of pups, live births, stillborn, post-natal loss, abnormal pups, body weight.

GROSS EXAMINATION OF DEAD PUPS:
no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Animals were euthanased when found to be moribund or when the adequate number oflitters according to guideline OECD 421 was reached.

- Maternal animals: Dams with offspring were sacrificed on Day 4 post-partum or shortly thereafter.

GROSS NECROPSY
- Gross necropsy consisted of: external and internal examinations including the cervical, thoracic, and abdominal viscera. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively: Epididymides, ovaries, testes and organs with macroscopic alterations. Additional findings in non-protocol organs present on slides were only recorded, if they were of pathological significance.
Testes and epididymes of all male adult animals were weighed. The ovaries, testes, epididymes, accessory sex organs and all other organs showing macroscopic lesions of all adult animals were preserved.
Statistics:
The arithmetic mean, standard deviation and median were calculated for all grouped numerical data originating from the monitoring of body mass and gross pathology (organ mass). In addition, the statistical software Graph Pad Prism for Mac, Version 5.01c. was used to calculate detailed column statistics (minimum/maximum data, 75% percentiles, std error, upper and lower confidence interval 95%). The study director pre-selected data sets of the ranked or incidental data for further analysis to those showing at least few alterations. Most statistical hypotheses in this study are characterised best as “many to one”– a vehicle control vs. three treatment groups. Therefore, the adequate analysis method is a One-Way ANOVA (Analysis of variance), followed by a post hoc t-test. With interval-scaled data, the One-Way ANOVA was supplemented by Dunnett’s post-hoc t-test. In case a Bartlett’s test for equal variances indicated that a data set may be heteroscedastic, it was analysed additionally by the Kruskal-Wallis test (rank transformation) and Dunn’s post-hoc t-test. However, the Study Director decided whether the additional test was necessary – data sets of single parameters were assessed as a whole, indications for heteroscedastic data subsets were disregarded. Ordinal-scaled data would have been analysed by the Kruskal-Wallis test, supplemented by Dunn’s t-test. The entire deductive statistics were performed using Graph Pad Prism. The significance level was set to 0.05.
Reproductive indices:
- Pre-implantation loss: corpora lutea - implantations
- Pre-natal loss: implantations - live births
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
all test groups: laboured breathing and wheezing breath sounds; 7 animals died due to test item reaching the respiratory tract, 1 animal died due to an application error
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
male high dose group: reduced mean body weight and reduced relative body weight gain; females high dose group: reduced relative body weight gain
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
male high dose group: reduced mean body weight and reduced relative body weight gain; females high dose group: reduced relative body weight gain
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
high dose group: mean numbers per dam of corpora lutea slightly and statistically significantly reduced; medium and high dose: weak evidence for a delayed conception
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Laboured breathing and vocalisations, in animals from all groups treated with test item probably due to the high surface activity of the test item, small amounts of test item solution reached the respiratory tract, some leading to fatalities by acute exogenous lipid pneumonia. Eight animals died spontaneously or were killed during the course of the study. Their premature death of most of these animals is considered to be most likely due to an inadvertent deposition of a small amount of the test item at the laryngeal orifice that was inhaled during inspiration. One rat was euthanised due to an application error.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In high dose males, body weight and relative body weight gain were prominently reduced throughout the entire in-life phase with a net mean body mass loss in the first two weeks of application. Females were less affected during pre-mating, and the effect was even less pronounced during the following weeks. The observed tendency of a lowered relative body weight gain in females of the high dose group during gestation and lactation reached the level of statistical significance in data from the day of birth.
A reduction of the mean relative food consumption as well as a strong increase of mean relative water consumption of males and females from the high dose groups was observed during the first two weeks of application (pre-mating). During the following weeks, mean relative food consumption was mildly (sires) or only in parts (dams Day 14, Day 20 of gestation, Day 0 of lactation) affected. However, the mean relative water consumption of males remained prominently raised.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
In the high dose group, the mean numbers per dam of corpora lutea were slightly and statistically significantly reduced. In the medium and high dose group a weak evidence for a delayed conception was apparent.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The organ mass gave no evidence for toxicological effects of the test item on the sexual organs of Wistar rats.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At gross necropsy, no abnormalities were found that could be related to the administration of the test item.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The histomorphological examination of selected rat organs of the male and female genital system (24 males and 24 females) did not reveal morphological lesions related to the test item.


Dose descriptor:
LOEL
Remarks:
reproduction
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: observed effects were considered secondary effects due to parental toxicity
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduction of mean relative food consumption and strong increase of mean relative water consumption, reduced mean body weight and body weight gain
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
high dose group: significantly raised post natal loss, reduced mean numbers of live pups
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduction of body mass with increasing dosage and a statistically highly significant reduction of mean pup body mass and mean litter mass in the high dose group at day four of lactation
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
In the high dose group significantly reduced mean numbers of live pups at Day 4 were observed. Data of the numbers of abnormal pups born, or the loss of offspring (pre-implantation and pre-natal) were normal for rats of this strain and age. In conclusion, post-natal loss was significantly raised in the high dose group.

BODY WEIGHT (OFFSPRING)
A tendency of a reduction of body mass for both genders with increasing dosage and a statistically highly significant reduction of mean pup body mass and mean litter mass in the high dose group at day four was apparent. A mild and statistically significant reduction of male mean pup body mass was found at day of birth in the high dose group when compared to the vehicle control.
Dose descriptor:
NOAEL
Remarks:
reproduction
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: observed effects are considered to be secondary effects due to parental toxicity
Dose descriptor:
LOAEL
Remarks:
reproduction
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: observed effects are considered to be secondary effects due to parental toxicity
Reproductive effects observed:
not specified

Table 1. Summarised results of the study.

OBSERVATIONS

Dosage (units)

 

 

Vehicle

 

 

Low dose

 

 

Medium dose

 

 

High dose

Pairs started

11*

11#

11#

11#

Totals after first and second mating

Females achieving pregnancy

10

8

9

11

Conceiving Days

1 - 5

10

8

6##

10

Conceiving days 6

0

0

1##

1

Pregnancy = 21 days

0

0

0##

0

Pregnancy = 22 days

8

4

5##

10

Pregnancy ≥ 23 days

2

4

2##

1

Dams with live young born

10

8

9

11

Dams with live young at Day 4 pp

10

8

8

11

Corpora lutea / dam (mean)

16,3

14,1

16,3

12,6

Implantations / dam (mean) 

12,6

9,4

12,6

11,5

Live pups / dam at birth (mean) 

10,7

7,9

9,6

9,6

Live pups / dam at day 4 (mean) 

10,1

6,9

8,9**

5,0

Litter mass Day 0 

64,8

50,3

59,8

48,9

Litter mass Day 4

92,6

67,8

91,4

42,2

No of pups

Live pups born Day 0 (count)

107

63

86

106

Stillborn (count) 

3

3

11

2

Total of pups born Day 0 (count) 

110

66

97

109

Stillborns / pups total (%)

2.8

4.8

12.8

2.8

Sex ratio

Sex ratio d0 (total numbers M / F

54 / 56

33 / 33

49 / 48

54 / 55

Sex ratio d0 (mean, M / F) 

1

1

1

1

Sex ratio d4 (total numbers M / F

56 / 45

31 / 24

38 / 33 **

30 / 25

Sex ratio d4 (mean, M / F) 

1.2

1.3

1.2

1.2

Body mass pups (g)

 

Male pups Day 0 (mean) 

6,1

 

6,3

5,8

5,2

Female pups Day 0 (mean) 

5,8

6,1

5,5

4,9

Male pups Day 4 (mean) 

9,6

10,3

9,6

7,7

Female pups Day 4 (mean) 

9,1

10,0

8,9

7,3

Abnormal pups 

 

 

 

 

Dams with 0 

10

8

9

11

Dams with 1 

0

0

0

0

Dams with ≥ 2 

0

0

0

0

Loss of offspring 

Pre-implantation (corpora lutea minus implant)        

Dams with pre-implantation loss (count) 

10

8

9

11

Pre-implantation loss (mean/group) 

3,7

4,8

3,9

2,4

Pre-natal (implantations minus live births) 

Dams with pre-natal loss (count) 

10

8

9

11

Pre-natal loss (mean/group) 

1,9

1,5

2,9

1,8

Post-natal (live births minus alive at post natal day 4)        

Dams with post-natal loss (count) 

10

8

9

11

Post-natal loss (mean pups/group) 

0.6

1.0

0.7

4.6

* female E112 invalidated

# one female each died within pre-mating phase;

## not reflecting data of two dams (no sperm plug found during mating phase, but pregnancy achieved)

** not reflecting litter mass data of one dam (E135) at Day 4 (data point lost)

Conclusions:
No effect on reproductive performance were observed.
Executive summary:

Considering the fact that no abnormalities were found by a gross necropsy and in the following histopathological examination of the reproductive organs, the authors concluded that the negative effects of the high-dose of the test item on Wistar rat reproduction were not caused by its toxic properties on those organs directly, but rather on other secondary local or systemic factors affecting parental animal health.

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data from handbook or collection of data. Peer-reviewed data. Only secondary source available.
Principles of method if other than guideline:
2-year chronic oral toxicity study in 200 rats.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: Albino Sherman Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 100-250 g
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
24 months
Frequency of treatment:
7 days/week
Dose / conc.:
1 000 other: mg/kg bw/day nominal in diet
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS: Yes (no further information)

BODY WEIGHT: Yes (no further information)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 30, Day 90, 6 months and 24 months
- Parameters: red and white blood cell count, haemoglobin content and differential count

Oestrous cyclicity (parental animals):
FERTILITY ASSESSMENT: Yes (no further information)
Sperm parameters (parental animals):
FERTILITY ASSESSMENT: Yes (no further information)
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes. Liver, spleen, heart, lungs, stomach, large intestine, small intestine, adrenal glands, gonads, pancreas and brain.
HISTOPATHOLOGY: Yes. Liver, spleen, heart, lungs, stomach, large intestine, small intestine, adrenal glands, gonads, pancreas and brain.
Rats were killed at 1, 3, 6 and 24 months for necropsy and tissues collected for microscopic examinations (no further information)
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY: At 1, 3 and 6 months, no significant differences were observed in mortality.

BODY WEIGHT AND WEIGHT GAIN: At 1, 3 and 6 months, no significant differences were observed in body weight gain.

HAEMATOLOGY: At 1, 3 and 6 months, no significant differences were observed in haematology.

GROSS PATHOLOGY: At 24 months, the only consistent difference that could be attributed to the test article was minor hyperplasia of the stratified squamous epithelium with excess keratin formation of the cardiac mucosa of the stomach in rats receiving the highest exposure to the test article (group 1: 2% in the diet after 6 months, group 3: 1% in the diet).
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Clinical signs:
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 ng/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The available information comprises adequate and reliable (Klimisch score 1 and 2) studies from the registered substance and a source substance with similar structures and intrinsic properties. Read-across is justified based on common precursors and metabolic degradation products and consistent trends in environmental fate, ecotoxicological and toxicological profile. The selected studies are thus sufficient to fulfil the standard information requirements set out in Annexes VIII - X, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

N-methyl-N-(C18-(unsaturated)alkanoyl)glycine was tested for toxicity to reproduction in a 28-day screening study according to OECD guideline 421 under GLP conditions (vivo Science, 2010). The dose was based on the results of a 28-day dose range finding study, with 3 animals per dose and sex orally exposed to 50, 250 and 1000 mg/kg bw/day. In the main study, groups of 12 Wistar rats (m, f) were given 50, 250 and 1000 mg/kg bw/day of the test material by gavage. Male animals were treated with the test material two weeks before mating, throughout the mating period and until the study was terminated. The female animals were exposed during 2 weeks before mating, up to 14 days until mating, an average of 21 days of gestation, and a minimum of 4 days of lactation. A concurrent negative control group receiving the vehicle (1% sodium carboxymethyl cellulose + 0,1% Polysorbate 80 (Tween 80), diluted in water) only was included in the testing as well.

Examination of the parental animals revealed laboured breathing and vocalisations, in animals from all groups treated with test item probably due to the high surface activity of the test item. Small amounts of test item solution reached the respiratory tract, some leading to fatalities by acute exogenous lipid pneumonia. Eight animals died spontaneously or were killed during the course of the study. Their premature death of most of these animals is considered to be most likely due to an inadvertent deposition of a small amount of the test item at the laryngeal orifice that was inhaled during inspiration. One rat was euthanised due to an application error. In high dose males, body weight and relative body weight gain were prominently reduced throughout the study with a net mean body mass loss in the first two weeks of application. In females of the high dose group, a significantly lowered relative body weight gain during gestation and lactation was observed. A reduction in the mean relative food consumption and an increase in the mean relative water consumption was observed in all animals from the high dose groups during the first two weeks of application. No effects on the organ mass of the sexual organs and no histomorphological effects on the genital system were observed. In addition, no test item related abnormalities were found during gross necropsy.

Regarding the reproductive performance, animals of the high dose groups showed a slightly but statistically significantly reduced mean number per dam of corpora lutea. Furthermore, in the medium and high dose group a weak evidence for a delayed conception was apparent. In the high dose group significantly reduced mean numbers of live pups at Day 4 were observed. However, the numbers of abnormal pups born and the pre-implantation and pre-natal loss were normal for rats of this strain and age. A statistically highly significant reduction of mean pup body mass and mean litter mass in the high dose group at day four was apparent. A mild and statistically significant reduced mean pup body mass (males) was found at day of birth in the high dose group when compared to the vehicle control as well. Considering the fact that no abnormalities were found by a gross necropsy and in the following histopathological examination of the reproductive organs of the parental animals, the authors concluded that the negative effects of the high dose of the test item on reproduction were not caused by its toxic properties on those organs directly, but rather on other secondary local or systemic factors affecting parental animal health. Thus, a systemic NOAEL for the parental animals (m, f) of 250 mg/kg bw/day and a NOAEL (m, f) for reproduction of 1000 mg/kg bw/day was evaluated in the study.

In addition, data from a 2-year study with 200 rats, treated with sodium N-lauroylsarcosinate (CAS 137-16-6) is considered. A fertility assessment was part of the chronic study (CIR, 2001). Groups of 25 Wistar rats per sex and dose were given 0.05, 0.2 and 1% of the test material in the diet, 7 days/week for 24 months. The concentration of the test material in the low dose group was increased to 2% (equiv. to 1000 mg/kg bw/day) after 6 months. A concurrent negative control group receiving the plain diet was included. At one, three and six months no significant differences were observed in mortality, body weight gain and haematology. At the end of the study period, the only consistent difference observed in the gross pathology was minor hyperplasia of the stratified squamous epithelium with excess keratin formation of the cardiac mucosa of the stomach in rats receiving the highest exposure to the test article (group 1: 2%; group 3: 1% in the diet). No details were given for fertility assessment; however the authors reported that the test material did not show any significant effects on reproductive function of the parental animals.

In summary, both the studies with N-methyl-N-(C18-(unsaturated)alkanoyl)glycine and the structurally related substance sodium N-lauroylsarcosinate showed no effects on reproductive performance and a NOAEL for reproductive toxicity of 1000 mg/kg bw/day was defined.

Effects on developmental toxicity

Description of key information

Developmental toxicity NOAEL >= 250 mg/kg bw/day (OECD 414, rats, GLP).

Developmental toxicity NOAEL >= 500 mg/kg bw/day (OECD 414, rabbits, GLP).

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
23 Nov 2016 -
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted Jan 2001
Deviations:
yes
Remarks:
(purity of the test material not given)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147
Version / remarks:
24 Nov 2000
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS
- Age at study initiation: 18 - 22 weeks
- Weight at study initiation: 2.31 - 4.36 kg
- Housing: The animals were individually housed during acclimatization and gestation period, and co-housed (M/F ration: 1/1) during mating period in suspended cages fitted with perforated floor panels. An aspen chew block was provided to each cage as environmental enrichment.
- Diet: Teklad 2930 pelleted diet, initially 150 g/animal/day during acclimatization up to one week prior to the onset of mating and 200 g/animal/day thereafter. In addition, a small supplement of autoclaved hay was given on a daily basis and a small amount of chopped fresh vegetables were given twice weekly.
Water: Potable water, ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 21
- Humidity (%): 45 - 70
- Photoperiod (hrs dark / hrs light): 10/14

IN-LIFE DATES: From: 23 Nov 2016 To:
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared weekly by dissolving appropriate amounts of the test material in purified water yielding a final concentration of 100 mg/mL. The remaining concentrations were prepared by serial dilution with further quantities of vehicle.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in water formulations at nominal concentrations of 2 mg/mL and 200 mg/mL was confirmed following refrigeration for 17 days at the same testing facility (Study No. KK40KJ)
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: yes
- Other: The day of mating is considered to be Day 0 of Gestation. Where possible only females mating at least twice were allocated.
Duration of treatment / exposure:
Day 6 - 28 of gestation
Frequency of treatment:
daily, 7 days/week
Duration of test:
Day 29 post mating
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were chosen based on the results of a preliminary study for effects on embryo-fetal development in the New Zealand White rabbit at the same testing facility (Study No. SF87BJ). In that study, treatment with the test substance at 125 or 250 mg/kg/day was well-tolerated, with no effects apparent that precluded the use of these doses on the subsequent main study for effects on embryo-fetal development. Treatment at 500 mg/kg/day elicited effects of reduced body weight gain and food consumption on the does, and reduced litter and fetal weights amongst the litters of females which received the test substance at 500 mg/kg/day, the magnitude of which did not preclude the use of this dose on the subsequent main study for effects on embryo-fetal development where the highest dose was chosen with the aim to induce some developmental and/or maternal toxicity. Doses of 125, 250 and 500 mg/kg/day were considered appropriate for the subsequent main study for effects on embryo-fetal development.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes, all animals were inspected visually for evidence of clinical signs and mortality.
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes, a detailed phyisical examination was performed on all animals to monitor general health.
- Time schedule: On Days 0, 6, 12, 18, 24 and 29 after mating

BODY WEIGHT: Yes, body weights of all animals were recorded.
- Time schedule for examinations: Weekly during acclimatizatuin, on the day of mating and on Days 0, 3 and 6 - 29 post mating.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 1 after mating.

POST-MORTEM EXAMINATIONS: Yes, all adult animals were subject to a detailed necropsy. Animals surviving until the end of the scheduled study period were killed on Day 29 post mating. Females that exhibited pregnancy loss were killed on the day of detection.
- Organs examined: All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes, for females surviving to term.
- Number of corpora lutea: Yes, for all animals.
- Number of implantations: Yes, for all animals.
- Number of early resorptions: Yes, for all animals.
- Number of late resorptions: Yes, for all animals.
Fetal examinations:
- External examinations: Yes, all fetuses were weighed, and were subject to an external examination and a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was recorded.
- Soft tissue examinations: Yes, serial sections were examined for soft tissue abnormalities.
- Skeletal examinations: Yes, the torsos of the decapitated fetuses and the remaining fetuses were eviscerated and fixed in Industrial Methylated Spirit.
- Head examinations: Yes, half per litter were decapitated and the heads were fixed in Bouin`s fluid.
Statistics:
The following data types were analysed at each timepoint separately:
- Body weight, using absolute values and gains over appropriate study periods
- Gravid uterine weight and adjusted body weight
- Food consumption, over appropriate study periods
- Litter size and survival indices
- Fetal, placental and litter weight

Detailed information on statistical information is provided under "any other information on materials and methods incl. tables".
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
125 mg/kg bw/d: 1/22 females had shown swaying and gasping respiration prior to death, and was found dead on Day 15 of gestation.
500 mg/kg bw/d: 1/22 females showed signs of respiratory distress and was getting very stressed when the technicians tried to touch her. The animal was despatched to necropsy on Day 9 of gestation (plase refer to "Mortality").
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Control: 1/22 females was found dead on Day 21 of gestation. The animal did not show any clinical signs prior to death, and there were no abnormalities detected at macroscopic examination.

125 mg/kg bw/d: 1/22 females was found dead on Day 15 of gestation. Clinical signs and a loss of body weight were observed prior to death. Macroscopic examination revealed abnormalities (please refer to "Gross pathological findings"). However, according to the study director the death was unclear and was not considered to be treatment-related. 1/22 females was despatched to necropsy on Day 24 of gestation following evidence of abortion. No clinical signs were observed prior to death and no abnormalities were detected at macroscopic examination.

500 mg/kg bw/d: 1/22 females was despatched to necropsy on Day 9 of gestation having shown signs of respiratory distress and a lost 410 g of body weight since the start of dosing. Macroscopic examination revealed abnormalitites (please refer to "Gross pathological findings"). According to the study director, the death was considered to be non-treatment related.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no clear effect of treatment on the body weight performance of females receiving the test substance at 125 or 250 mg/kg bw/d when compared with that of the control group. The mean body weight loss of females receiving dosed at 500 mg/kg bw/d was slightly greater than that of the control group after the start of treatment (between Days 6 and 8 after mating), and body weight gain was slightly less than that of the control animals from Days 8 - 29 after mating.
When mean values of body weight and body weight gain were adjusted for the contributions of the gravid uterus, overall maternal mean body weight loss during Days 6 - 29 of gestation was greater at 500 mg/kg bw/d than in the control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There was no clear effect of treatment on the food consumption of females receiving the test substance at 125 or 250 mg/kg bw/d when compared with that of the control group. The food consumption of females dosed at 500 mg/kg bw/d was slightly lower than that of the control group from the start of treatment (Day 6 after mating), resulting in a lower overall (Day 6 - 28 after mating) food consumption.
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, non-treatment-related
Description (incidence and severity):
125 mg/kg bw/d: The macroscopic examination of the animal found dead on Day 15 of gestation, revealed discoloured (dark) adipose tissue in the thoracic region, dark staining to the muzzle, dark fluid in the nasal turbinates, and dark right lungs.
500 mg/kg bw/d: The animal which was despatched to necropsy on Day 9 of gestation had poorly defined, dark areas on the lungs, gas in the jejunum and duodenum, and findings in the stomach consisting of a raised and firm antrum with a depressed area on the glandular mucosa at necropsy.

These findings were not considered to be treatment related or toxicologically relevant.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
125 mg/kg bw/d: The animal which was despatched to necropsy on Day 24 of gestation following evidence of abortion, was confirmed as pregnant with 7 implantations, 5 of which were early resorptions, 1 late resorption and 1 aborted implantation. However, according to the study director, this single abortion is considered not to be related to treatment.
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 examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
2/22 females of the control group and of the highest dose group, respectively, 1/22 females of the low-dose group and 3/22 females of the mid-dose group were not pregnant.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse maternal toxicity was observed at the highest tested dose level.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
500 mg/kg bw/d: Fetal weights were marginally lower than the fetal weight of the control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
500 mg/kg bw/d: Litter weights were marginally lower than the fetal weight of the control group.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
500 mg/kg bw/d: There were two minor fetal pathology findings including a slightly high incidence of additional cranial sutures, and a slightly high incidence of delayed ossification of the cervical vertebrae. According to the study director, the slightly high incidence of additional cranial sutures is in isolation considered not to represent an adverse effect of treatment as cranial bone development was normal. The delayed ossification of the cervical vertebrae represents the results of an evaluation at a snapshot in time (ossification would be expected to continue as the animals matured) and may be linked to the marginally lower mean fetal weights. The incidence of 7th costal cartilage not connected to sternum was lower compared to concurrent controls. According to the study director, the values were slightly high in the control and low-dose group, respectively, and slightly low in the high-dose group when compared with historical control data, thus the finding was considered to be not toxicologically significant.
Visceral malformations:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse embryo-fetal toxicity was observed at the highest tested dose level.
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: skull sutures
skeletal: vertebra
Description (incidence and severity):
Two minor fetal pathology findings including a slightly high incidence of additional cranial sutures, and a slightly high incidence of delayed ossification of the cervical vertebrae were observed at 500 mg/kg bw/d. According to the study director, the slightly high incidence of additional cranial sutures is considered not to represent an adverse effect of treatment as cranial bone development was normal. The delayed ossification of the cervical vertebrae represents the results of an evaluation at a snapshot in time (ossification would be expected to continue as the animals matured) and may be linked to the marginally lower mean fetal weights.
Key result
Developmental effects observed:
no
Conclusions:
Under the conditions of the study the test substance did not induce any treatment-related biologically relevant malformations in the developing unborn organism in the presence of maternal toxicity. Therefore, the test substance does not meet the criteria for classification for prenatal developmental toxicity according to Regulation (EC) No 1272/2008. The available data is thus conclusive but not sufficient for classification.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
04 Aug - 05 Dec 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 22 January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted August 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSa No 8147, 24 November 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD® (SD)IGS BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Margate, UK
- Age at study initiation: not specified in report, animals purchased time-mated
- Weight at study initiation: 197-268 g (females on arrival, prior to GD3)
- Housing: individual in solid-floor propylene cages with stainless steel lids in a single air-conditioned room, bedding with softfood flakes
- Diet: pellet diet (Rodent 2018C Teklad Global Certified Diet, Harlan; Oxon; UK) ad libitum, environmental enrichment provided in the form of wooden chew blocks and cardboard fun tunnels
- Water: ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 04 Aug 2013 (first day of treatment) To: 22 Aug 2013 (final day of necropsy)
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
the test item was prepared at the appropriate concentrations as a solution in distilled water.

VEHICLE
- Concentration in vehicle: 6, 20 and 50 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by high performance liquid chromatography (HPLC/UV) using an external standard technique.The formulations investigated during the study were found to comprise test item in the range of 101% to 104% and, thus, the required content limit ± 10% with reference to the nominal content was met. The test item was found to be stable in the formulations when kept for twenty one days in the refrigerator due to results which met the variation limit of 10% from the time-zero mean. Thus, the results indicate the accurate use of the test item and distilled water as vehicle during this study. The formulations were found to be homogeneously prepared, and sufficient formulation stability under storage conditions was approved.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
Gestation Day 5-19
Frequency of treatment:
daily, 7 days/week
Duration of test:
until Gestation Day 20 (terminal sacrifice)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 P females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on previous toxicity data.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked included: following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioural changes once daily during the gestation period. An additional observation was also performed five hours after dosing during the normal working week. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: individual body weights were recorded for each surviving individual animal at Day 3, 5, 6, 7, 8, 11, 14, 17 and 20 (termination kill) of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined: Yes, at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily by visual inspection of water bottles
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: ovaries and uteri of pregnant females, full external and internal examination; any macroscopic abnormalities were recorded; the stomach was retained from all females
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: fetal sex, placental weight, position and type of intrauterine implantation
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: No
Statistics:
Group mean values were calculated to include data from all females with live fetuses on Day 20 of gestation. As the litter was the standard unit of assessment, values were first calculated within the litter, and group mean values represent the mean of these individual litter values.
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Bartlett´s test for homogeneity of variance and one way analysis of variance, followed by Dunnett´s multiple comparison test or, if unequal variances were observed, an alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.
Probability values (p) demonstrating different levels of significance were chosen as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p>=0.05 (not significant)
Indices:
Pre implantation loss: [(number of corpora lutea-number of implantations) / number of corpora lutea] x 100;
Post-implantation loss: [(number of implantations–number of live foetuses)/number of implantations] x 100;
Sex ratio: % male fetuses (sex ratio)= [Number of male foetuses / Total number of foetuses] x 100

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
250 mg/kg bw/day: the female found dead on Day 10 did not show any clinical signs prior to Day 10. The female found dead on Day 18 had noisy respiration on Days 10 and 17 and increased salivation and pilo-erection on Day 17.
5/22 females showed incidences of increased salivation between Days 10 and 19 and 3/22 females also showed noisy respiration on either Day 6 or 7.

100 mg/kg bw/day: 1/24 females showed increased salivation on Day 18 and 1/24 females had noisy respiration on Day 13.

Observations of this nature are commonly observed following the oral administration of an unpalatable or irritant test item formulation and are considered not to represent true systemic toxicity.

Control: 1/24 females had fur loss on Day 18. In the absence of treatment this was considered of no toxicological importance.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
250 mg/kg bw/d: One female treated was found dead on Day 10 and another female was found dead on Day 18.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
250 mg/kg bw/day: body weight gain of the females was lower throughout the treatment period and cumulative body weight gain to Day 14 (where body weight is not greatly influenced by the weight of the gravid uterus) was 24% lower than the corresponding control value. By termination on Day 20, cumulative body weight gain was 15% lower than the corresponding control values. When final body weight was adjusted for the gravid uterus, an overall body weight gain reduction was evident during gestation.

100 mg/kg bw/day: females showed a slight reduction in cumulative body weight gain from Day 7 onwards. When final body weight was adjusted for the gravid uterus, an overall body weight gain reduction was evident during gestation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
250 mg/kg bw/day: females showed a reduction in food consumption throughout the treatment period. Statistically significant reductions were evident between Days 8 and 11 (p < 0.001) and Days 14-17 (p< 0.05) compared with the control group.
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:
not examined
Description (incidence and severity):
The female treated with 250 mg/kg bw/day that was found dead on Day 10 had gaseous distention in the stomach and gastro-intestinal tract. The other interim death female had sloughing on the non-glandular region of the stomach, seven dead foetuses in the right uterine horn and five dead foetuses in the left uterine horn. The remaining females treated with 250 mg/kg bw/day that were terminated on Day 20 all had sloughing on the non-glandular region of the stomach.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
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 examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Females from all treatment groups showed a statistically significant reduction (p<0.05 - p<0.01) in the number of corpora lutea. The intergroup differences did not show a true dose related response and were considered to be incidental and unrelated to treatment due to ovulation and mating occurring prior to the administration of the test item. As a consequence of incidentally lower number of corpora lutea in treated females litter size was statistically significantly reduced (p<0.05) in all treated females. The intergroup differences did not show a true dose related response and in the absence of any effect on post-implantation loss and mean fetal weights it supports the assumption that the intergroup differences in litter size was considered to be incidental and of no toxicological importance.
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was no obvious adverse effect of maternal treatment on litter data as assessed by the mean number of implantations, early and late embryonic/fetal deaths and live fetuses or sex ratio, as assessed by percentage male.
For all dose groups, there were no significant treatment-related trends in the proportion of fetuses (or litters) with evidence of visceral or skeletal anomalies. The types of external, visceral and skeletal anomalies were those commonly observed for this type of study. There were no findings that were considered to represent any known malformations.
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse effect on developmental toxicity observed at the highest tested dose level.
Abnormalities:
not specified
Developmental effects observed:
not specified

All treated females showed statistically significant reduction of corpora lutea and in consequence reduced litter sizes. The intergroup differences did not show a true dose related response and were considered to be incidental and unrelated due to ovulation and mating occurring prior to the administration of the test item starting on GD5. The absence of any effect on post-implantation loss and mean fetal weights supports the assumption that these intergroup differences in litter size were considered to be incidental and of no toxicological importance. Foetuses of the 250 mg/kg bw/day group showed a statistically significant increase in the percent of foetuses showing one or more ossified forepaw phalanges. Due to the isolated occurrence of this observation it is not considered to be a true developmental effect and it is therefore of no biological importance.

Table A: Group Mean Litter Data Values (Mean ± SD)

Dose Level (mg/kg bw/day)

Number of Litters

Number of Corpora Lutea

Number of Implants

Number of Embryonic/Fetal Deaths

Number of Live Implants

Early

Late

Total

Male

Female

Total

0 (control)

23

15.2 ± 1.8

13.9 ± 1.6

0.1 ± 0.3

0.1 ± 0.6

0.3 ± 0.7

6.9 ± 1.8

6.8 ± 1.9

13.7 ± 2.1

30

21

14.3* ± 1.8

13.1 ± 2.0

1.0 ± 2.4

0.1 ± 0.4

1.0 ± 2.5

6.0 ± 2.7

6.1 ± 2.4

12.1* ± 3.4

100

24

13.7* ± 1.9

13.0 ± 1.8

0.6 ± 1.3

0.2 ± 0.7

0.8 ± 1.4

6.5 ± 2.1

5.6 ± 1.7

12.1* ± 2.5

250

22

13.9** ± 1.2

13.2 ± 1.1

0.5 ± 1.4

0.2 ± 0.9

0.7 ± 1.6

6.5 ± 2.0

6.0 ± 1.9

12.5* ± 2.2

p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 not significant

Table A: Group Mean Litter Data Values (continued) (Mean ± SD)

Dose Level (mg/kg bw/day)

Implantation Loss (%)

Male foetuses (%)

Mean Male Fetal Weight (g)

Mean Female Fetal Weight (g)

Mean Fetal Weight (g)

Mean Placental Weight (g)

Litter Weight (g)

Total Placental Weight (g)

Pre

Post

0 (control)

8.4 ± 7.4

2.3 ± 7.2

50.4 ± 11.3

4.117 ± 0.260

3.935 ± 0.264

4.026 ± 0.257

0.564 ± 0.047

54.883 ± 8.672

7.667 ± 1.141

30

8.0 ± 10.0

8.4 ± 19.4

48.6 ± 15.2

4.152 ± 0.405

3.881 ± 0.372

4.005 ± 0.365

0.578 ± 0.155

48.345 ± 13.761

6.729 ± 1.942

100

5.0 ± 5.2

6.9 ± 12.2

53.7 ± 12.8

4.166 ± 0.339

3.886 ± 0.269

4.036 ± 0.289

0.542 ± 0.060

49.142 ± 11.318

6.540 ± 1.503

250

4.8 ± 5.3

5.5 ± 12.5

52.2 ± 13.7

4.221 ± 0.347

4.033 ± 0.327

4.126 ± 0.320

0.540 ± 0.057

51.237 ± 8.200

6.744 ± 1.369

 

Table B: Group Mean Cumulative Body Weight Change Values (Mean ± SD)

Dose Level (mg/kg bw/day)

Number of Animals

Cumulative Body Weight Change (g) from GD5

GD6

GD7

GD8

GD11

GD14

GD17

GD20

0 (control)

23

4.0 ± 3.4

7.6 ± 3.7

13.0 ± 5.1

33.6 ± 6.6

51.6 ± 8.1

82.3 ± 10.4

127.8 ± 14.9

30

21

5.1 ± 5.4

7.1 ± 8.2

12.9 ± 10.2

34.0 ± 12.9

53.5 ± 15.5

82.2 ± 19.2

127.0 ± 23.4

100

24

2.3 ± 5.3

6.1 ± 5.5

8.8 ± 7.5

27.6 ± 9.9

44.8 ± 13.8

74.2* ± 12.4

114.3* ± 17.2

250

24/22+

0.5 ± 9.0

3.6 ± 10.1

7.3 ± 11.2

21.7** ± 14.6

39.3* ± 17.8

68.2** ± 20.0

109.1** ± 24.7

+ = n=23 on GD10, n=22 on GD18
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 not significant

 

Table C: Group Mean Gravid Uterus Weight , Adjusted Body Weight and Body Weight Change Values (Mean ± SD)

Dose Level (mg/kg bw/day)

Number of Animals

Body Weight (g) on Days of Gestation

Body Weight Change (g) during GD5-20

Gravid Uterus Weight (g)

Adjusted Body Weight (g) GD20

Adjusted Body Weight (g) Change GD5-20

GD5

GD20

0 (control)

23

262.6 ± 20.6

390.4 ± 29.8

127.8 ± 14.9

84.663 ± 12.502

305.8 ± 23.4

43.2 ± 8.2

30

21

261.7 ± 12.9

288.7 ± 27.3

127.0 ± 23.4

75.544 ± 18.733

313.2 ± 28.6

51.5 ± 22.7

100

24

260.6 ± 14.4

374.9 ± 24.8

114.3** ± 17.2

75.893 ± 15.615

299.0 ± 20.7

38.4* ± 14.6

250

24/22+

257.6 ± 15.5

368.1 ± 32.4

109.1** ± 24.7

77.535 ± 11.935

290.6 ± 25.0

31.6** ± 18.7

+ = n=23 on GD10, n=22 on GD18
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 not significant

 

Table D: Group Mean Food Consumption Values (Mean ± SD)

Dose Level (mg/kg bw/day)

Number of Animals

Food Consumption (g/rat/day) between Days of Gestation

3-5

5-8

8-11

11-14

14-17

17-20

0 (control)

23

23.0 ± 3.1

25.1 ± 2.5

26.5 ± 2.6

26.1 ± 2.8

25.9 ± 3.0

28.0 ± 2.4

30

21

23.6 ± 3.7

24.5 ± 3.9

27.1 ± 3.7

26.5 ± 3.7

26.0 ± 4.2

28.7 ± 3.8

100

24

22.8 ± 2.9

23.9 ± 2.8

24.9 ± 3.3

24.6 ± 3.7

24.5 ± 3.4

27.5 ± 2.9

250

24/22+#

22.3 ± 3.4

22.4 ± 5.3

21.5*** ± 4.7

23.3 ± 4.3

22.6* ± 4.2

25.4 ± 3.8

+ = n=23 on GD10, n=22 on GD18
# = Food consumption not performed on GD 8 and 11 for female Nos. 85 to 90 due to technician error
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 not significant

In conclusion the oral administration of the test substance to pregnant rats by oral gavage during gestation at dose levels of 30, 100 and 250 mg/kg bw/day resulted in treatment related effects detected in females treated with 250 and 100 mg/kg bw/day. The NOAEL was therefore considered to be 30 mg/kg bw/day.

No toxicological significant changes were detected in the offspring parameters measured. The NOEL for developmental toxicity was therefore considered to be 250 mg/kg bw/day. 

Conclusions:
Under the conditions of the study the test substance did not induce any treatment-related biologically relevant malformations in the developing unborn organism in the presence of maternal toxicity. Therefore, the test substance does not meet the criteria for classification for prenatal developmental toxicity according to Regulation (EC) No 1272/2008. The available data is thus conclusive but not sufficient for classification.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises adequate and reliable (Klimisch score 1) studies from a source substance with similar structures and intrinsic properties. Read-across is justified based on common precursors and metabolic degradation products and consistent trends in environmental fate, ecotoxicological and toxicological profile. The selected studies are thus sufficient to fulfil the standard information requirements set out in Annexes VIII - X, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Additional information

Data on developmental toxicity / teratogenicity are available for the structuraly analogue source substance sodium N-lauroylsarcosinate (CAS 137-16-6). Studies have been performed with rats as well as rabbits.

Rat

The test item sodium N-lauroylsarcosinate (CAS 137-16-6) was administered by gavage to three groups of time-mated pregnant Sprague-Dawley rats, between Days 5 and 19 of gestation, at dose levels of 30, 100, and 250 mg/kg bw/day (Harlan, 2014). A further group of time-mated pregnant females was exposed to the vehicle only (control).

Clinical signs, body weight change, food and water consumptions were monitored during the duration of the study.

At sacrifice (at Day 20 of gestation) all females were subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weight, foetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

With regard to maternal toxicity one female treated with 250 mg/kg bw/day was found dead on day 10 of gestation. Another female from this treatment group was found dead on Day 18 of gestation. There were no further unscheduled deaths.

Surviving females treated with 250 mg/kg bw/day showed incidences of increased salivation and noisy respiration during the treatment period. One female treated with 100 mg/kg bw/day also showed increased salivation on Day 18 of gestation and a further female showed noisy respiration on Day 13. No such effects were detected in females treated with 30 mg/kg bw/day.

The female found dead on Day 10 did not show any clinical signs prior to Day 10. The female found dead on Day 18 had noisy respiration, increased salivation and pilo-erection.

Females treated with 250 mg/kg bw/day and to a lesser extent females treated with 100 mg/kg bw/day showed a reduction in body weight development throughout the treatment period. Body weight gain adjusted for gravid uterus weight between Days 5 and 20 was also redued in these females. No such effects were detected in females treated with 30 mg/kg bw/day.

Correspondingly, females treated with 250 mg/kg bw/day also showed a reduction in food consumption throughout the treatment period. No such effects were detected in females treated with 100 or 30 mg/kg bw/day.

With regard to developmental toxicity no toxicologically significant effects were detected in the uterine parameters examined, in foetal viability or in growth and development. In addition, no treatment-related effects were detected on foetal external findings. No treatment-related effects were detected on skeletal development or in the type and incidence of skeletal or visceral findings in foetuses from females treated with 250, 100 or 30 mg/kg bw/day. Furthermore there were no findings considered to represent any known malformations.

In conclusion, as the oral administration of the test item to pregnant rats by gavage during gestation at dose levels of 30, 100 and 250 mg/kg bw/day resulted in substance-related effects in females treated with 250 and 100 mg/kg bw/day, the NOAEL for maternal toxicity was therefore considered to be 30 mg/kg bw/day.

No toxicological significant changes were detected in the offspring parameters measured. The NOAEL for reproductive and developmental toxicity was therefore considered to be 250 mg/kg bw/day, corresponding to the hightest dose tested.

Rabbit

Three groups of 22 females received the test item Sodium N-lauroylsarcosinate (CAS 137-16-6) at doses of 125, 250 or 500 mg/kg/day by oral gavage administration, from Day 6 to 28 after mating (Envigo, 2017). A similarly constituted control group received the vehicle, purified water at the same volume dose as treated groups. Animals were killed on Day 29 after mating for reproductive assessment and fetal examination. Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailedinternal visceral examination of the head or skeletal examination.

With regard to maternal toxicity one female of the control group and one female each of the mid- and high-dose group died during the study period. However, the deaths were not considered to be treatment-related, since no treatment-related clinical signs were observed after treatment with the test substance at 125, 250 or 500 mg/kg bw/d. In addition, there was no clear effect of treatment on the body weight or food consumption performance of females receiving Sodium N-lauroylsarcosinate (CAS 137-16-6) at 125 or 250 mg/kg bw/d when compared with that of the control group. The mean body weight loss of females exposed to 500 mg/kg bw/d was slightly greater than that of the control group after the start of treatment (between Days 6 and 8 after mating), and body weight gain was slightly lower than that of the controls from Days 8-29 after mating. The food consumption of females treated with 500 mg/kg bw/d was slightly lower than that of the Control from the start of treatment (Day 6 after mating), resulting in a lower overall (Day 6 - 28 after mating) food consumption.

When mean values of body weight and body weight gain were adjusted for the contributions of the gravid uterus, overall maternal mean body weight loss during Days 6-29 of gestation was greater than in controls at 500 mg/kg bw/d. There were no test item-related macroscopic abnormalities detected among the females at scheduled termination on Day 29 after mating.

Embryo-fetal survival was unaffected by treatment at 125, 250 or 500 mg/kg bw/day with mean numbers of implantations, resorptions, live young and percentages of sex ratio and pre and post-implantation loss being similar to Control values.

Mean placental, litter and fetal weights at 500 mg/kg bw/day were marginally lower (ca 10%) than controls.

Mean placental, litter, and male, female and overall fetal weights at 125 or 250 mg/kg bw/day were similar to the control group and unaffected by treatment.

At 500 mg/kg bw/d there was a slightly higher incidence of the minor fetal abnormality additional cranial sutures, which was outside Historical Control Data (HCD).An increase was also seen in the incidence of 20 thoracolumbar vertebrae, however this was within HCD. At 500 mg/kg bw/day there was also a slightly higher incidence of delayed ossification of the cervical vertebrae but this was considered to be related to the lower mean fetal weights seen in this group.

In conclusion, at 500 mg/kg bw/d, maternal body weight performance and food consumption and mean fetal weights were slightly reduced. Embryo-fetal survival was unaffected by treatment and fetal development was not adversely affected. The No-Observed-Adverse-Effect-Level (NOAEL) for maternal and embryo-fetal toxicity was concluded to be 500 mg/kg bw/day, corresponding to the highest dose tested.

Conclusion

Based on the reliable and adequate studies with the structural analogue source target Sodium N-lauroylsarcosinate (CAS 137-16-6) in two rodent species, no effects on maternal and embryio-fetal toxicity are anticipated for N-methyl-N-(C18-(unsaturated)alkanoyl)glycine.

Justification for classification or non-classification

Based on read-across from the structurally related substance Sodium N-lauroylsarcosinate and substance specific data, the available data on reproductive and developmental toxicity do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.