N-methyl-N-[C18-(unsaturated)alkanoyl]glycine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Screening (OECD 421, rat): NOAEL (reproduction) = 1000 mg/kg bw/day

An extended one-generation reproductive toxicity study according to OECD guideline 443 is proposed. When study results are available, the toxicity to reproduction endpoint will be re-assessed.

Link to relevant study records

Reference

Reference 1

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

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 or test system 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.

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study planned

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS
NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: N-methyl-N-[C18-(unsaturated)alkanoyl]glycine

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION
- Available GLP studies:
With respect to reproductive toxicity, only a screening study for reproductive / developmental toxicity according to OECD guideline 421 is available with the registered substance. In this study, no relevant effects in relation to reproductive / developmental toxicity have been observed. In addition, the available subchronic (90-day) oral repeated dose toxicity study and the pre-natal developmental toxicity study in rats, both conducted with the registered substance, as well as a pre-natal developmental toxicity study in rabbits performed with an adequate analogue substance, did not reveal any observations in reproductive tissues and organs indicative of reproductive toxicity.
- Available non-GLP studies:
No study to satisfy the information requirement of Annex X, Section 8.7.3., of the REACH Regulation (EC) No. 1907/2006 with the registered substance is available.
- Historical human data:
The registrant is not aware of any relevant human data suitable to satisfy the information requirement of Annex X, Section 8.7.3., of the REACH Regulation (EC) No. 1907/2006 with the registered substance is available.
- (Q)SAR:
No QSARs are available to reliably predict the endpoints assessed in an extended one-generation reproductive toxicity study (OECD 443). Therefore, QSAR assessment is not possible.
- In vitro methods:
No in vitro methods are available to reliably predict the endpoints assessed in an extended one-generation reproductive toxicity study (OECD 443). Therefore, in vitro studies are not possible.
- Weight of evidence:
No adequate and reliable data is available that possibly could be used for a Weight-of-Evidence assessment of reproductive toxicity for the registered substance.
- Grouping and read-across:
Grouping and read-across have been assessed thoroughly and a number of structurally similar substances sharing similar properties with respect to e-fate, ecotoxicology and toxicology have been identified. In fact, a robust read-across approach is applied for pre-natal development in rabbits. However, no adequate and reliable studies concerning reproductive toxicity are available in the data pool of suitable read-across source substances. In consequence, the only way to comply with the standard information requirement of Annex X, Section 8.7.3., of the REACH Regulation (EC) No. 1907/2006 is to perform an extended one-generation reproductive toxicity study according to OECD guideline 443 with the registered substance.
- Substance-tailored exposure driven testing:
The registered substance is used in a variety of applications. None of the conditions and prerequisites of Annex XI, Section 1.3., of the REACH Regulation (EC) No. 1907/2006 can be met. Therefore, substance-tailored exposure driven testing cannot be applied for the registered substances.
- Approaches in addition to above:
Not applicable
- Other reasons:
Not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
Annex X, Section 8.7. (Reproductive Toxicity), Column 2, of the REACH Regulation (EC) No. 1907/2006 stipulates the following specific adaption options:
‘The studies need not be conducted if:
— the substance is known to be a genotoxic carcinogen and appropriate risk management measures are implemented, or
— the substance is known to be a germ cell mutagen and appropriate risk management measures are implemented, or
— the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and there is no or no significant human exposure.
If a substance is known to have an adverse effect on fertility, meeting the criteria for classification as toxic for reproduction category 1A or 1B: May damage fertility (H360F), and the available data are adequate to support a robust risk assessment, then no further testing for fertility will be necessary. However, testing for developmental toxicity must be considered. If a substance is known to cause developmental toxicity, meeting the criteria for classification as toxic for reproduction category 1A or 1B: May damage the unborn child (H360D), and the available data are adequate to support a robust risk assessment, then no further testing for developmental toxicity will be necessary. However, testing for effects on fertility must be considered.’
None of the above mentioned conditions are met. Therefore, the specific Column 2 adaption option are not adequate to generate the necessary information for the registered substance.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed:
The testing proposal is based the results of a screening study for reproductive / developmental toxicity according to OECD guideline 421 and the lack of relevant effects in reproductive tissues and organs indicative of reproductive toxicity observed in subchronic (90-day) oral repeated dose toxicity (OECD 408) and pre-natal developmental toxicity studies (OECD 414).
The following study is proposed: Extended one-generation reproductive toxicity study according to Annex X, Section 8.7.3., of the REACH Regulation (EC) No. 1907/2006 (B.56 of the Commission Regulation on test methods or OECD guideline 443) in rats, oral route with the registered substance, specified as follows:
- Ten weeks premating exposure duration for the parental (P0) generation
- Dose level setting shall aim to induce some toxicity at the highest dose level
- Cohort 1A (Reproductive toxicity)
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation
For a justification of the test design, please refer to ‘Justification of study design’ below.

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals:
Ten weeks premating exposure duration for the parental (P0) generation as recommended by ECHA. This is the default value as specified by ECHA in recent decisions on OECD 443 studies.
- Basis for dose level selection:
The dose level setting shall aim to induce some toxicity at the highest dose level. Recently performed subchronic (90-day) oral repeated dose toxicity (OECD 408) and pre-natal developmental toxicity (OECD 414) studies with the registered substance indicated that substantial (maternal) toxicity was induced at dose levels of 1000 and 750 mg/kg bw/day. In the subchronic (90-day) oral repeated dose toxicity study, the initially chosen dose level of 1000 mg/kg bw/day had to be reduced to 600 mg/kg bw/day in the course of the study. Test animals tolerated the reduced dose level although toxicity was still observed.
- Inclusion/exclusion of extension of Cohort 1B:
This cohort is a standard requirement of an OECD guideline 443 study. Cohort 1B (Reproductive toxicity) will be included without extension to mate the Cohort 1B animals to produce the F2 generation.
Based on the available screening study for reproductive / developmental toxicity (OECD 421), subchronic (90-day) oral repeated dose toxicity study (OECD 408) and pre-natal developmental toxicity study in rats (OECD 414), all conducted with the registered substance, as well as the pre-natal developmental toxicity study in rabbits (OECD 414) performed with an adequate analogue substance, no relevant effects or adverse changes in reproductive tissues and organs indicative for reproductive / developmental toxicity have been observed. Therefore, the extension to mate the Cohort 1B animals is not justified.
- Termination time for F2:
Not relevant as the production of the F2 generation is not planned.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B:
The Cohorts 2A and 2B are not planned to be included in the study design. There are no adverse effects in relation to reproductive / developmental toxicity observed in any of the studies mentioned above which might trigger the inclusion of Cohort 2A and 2B.
- Inclusion/exclusion of developmental immunotoxicity Cohort 3:
The Cohort 3 is not planned to be included in the study design. There are no adverse effects in relation to reproductive / developmental toxicity observed in any of the studies mentioned above which might trigger the inclusion of Cohort 3.
- Route of administration:
The standard route of administration is oral via gavage. All previously conducted studies with repeated dose administration used this route of administration.
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals:
The standard species is the rat. It is intended to use the same rat strain as used in previous studies (Sprague-Dawley or Wistar) depending on the historical control database in the selected test facility. All other parameters will be selected as recommended in OECD guideline 443.

Species:

rat

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable (Klimisch score 1) study with the registered substance. Moreover, an extended-one generation reproductive toxicity study (OECD 443) is proposed. The study and testing proposal are thus sufficient to fulfil the standard information requirements set out in Annex X, Section 8.7.3., of Regulation (EC) No. 1907/2006 (REACH).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Screening study for reproductive / developmental toxicity

N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC No. 701-177-3) 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 only (1% sodium carboxymethyl cellulose + 0,1% Polysorbate 80 (Tween 80), diluted in water), 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. The 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.

Extended one-generation reproductive toxicity study

Based on the provisions of Annex X, Section 8.7.3., of Regulation (EC) No. 1907/2006 (REACH), an extended one-generation reproductive toxicity study according to OECD guideline 443, in rats, oral route with the registered substance is proposed. Once study results are available, the toxicity to reproduction endpoint will be re-assessed.

Effects on developmental toxicity

Description of key information

Pre-natal development (OECD 414, rat): NOAEL (maternal toxicity) = 300 mg/kg bw/day, NOAEL (development) = 300 mg/kg bw/day

Data obtained with N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC No. 701-177-3)

 

Pre-natal development (OECD 414, rabbit): NOAEL (maternal toxicity) = 500 mg/kg bw/day, NOAEL (development) = 500 mg/kg bw/day

Data obtained with analogue source substance sodium [dodecanoyl(methyl)amino]acetate (CAS No. 137-16-6)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Jan - 12 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 62 - 69 days
- Weight at study initiation: 215.8 - 273.8 g
- Housing: individually in MAKROLON cages (type III plus) with granulated textured wood bedding (Granulat A2, J. Brandenburg, Goldenstedt/Arkeburg, Germany)
- Diet: Ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light):12 / 12

IN-LIFE DATES: From: 13 Jan To: 11 Feb 2020

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous hydroxypropylmethylcellulose gel

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared daily by dissolving appropriate amounts of the test material in 0.5% aqueous hydroxypropylmethylcellulose gel. Dosing solutions were administered within 4 h after preparation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance is poorly water soluble. Therefore, an aqueous suspension of 0.5% hydroxypropylmethylcellulose gel was used as vehicle.
- Amount of vehicle: 10 mL/kg bw/day
- Lot/batch no.: 18D04-B03
- Supplier: Fagron Services B.V, Uitgeest, The Netherlands

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC with UV detection was employed to quantify the test item in the dose formulations. The analytical method was validated by LPT and reported in LPT Study No. 37647.
The homogeneity of the dose formulations was assessed on 21 and 24 January 2020 at the start of treatment, during (middle) administration and before administration to the last animal of the test item group. The concentration was analysed on 4 February 2020 during treatment before administration to the last animal of the group. The range of % nominal concentration was 93.8 - 105.9% for the homogeneity and 95.0 - 103.5% for concetration analysis.
In the method validation, also a stability assessment of the dose formulations was conducted. The samples of all storage conditions (24 h at room temperature and 7 or 64 days at -20 °C) met the acceptance criteria.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight, every night with the same male and female until proof of pregnancy was observed
- Verification of same strain and source of both sexes: yes, only sexually mature ('proved') male rats of the same breed served as partners.
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy

Duration of treatment / exposure:

Day 6 to 20 of gestation

Frequency of treatment:

daily, 7 days/week

Duration of test:

15 days, until Day 20 of gestation

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Dose reduction to 750 mg/kg bw/day due to one mortality, poor health and pronounced reduction of body weight for several animals.

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Remarks:

Dose reduction from 1000 mg/kg bw/day due to one mortality, poor health and pronounced reduction of body weight for several animals.

No. of animals per sex per dose:

25 P females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a foregoing range finding study, in which the test substance was administered orally to female rats at dose levels of 100, 300, 1000 mg/kg bw/day from Day 6 to Day 20 of pregnancy (LPT Study Report No. 37716). Adverse effects of reduced body weight, body weight gain and food consumption of the dams and reduced fetal weight and placental weight as secondary effects due to maternal toxicity were observed at 1000 mg/kg bw/day. Therefore, 100, 300 and 1000 mg/kg bw/day were selected as the dose levels for the main study.
- Fasting period before blood sampling for dam thyroid hormones: overnight
- Time of day for dam blood sampling: in the morning of day of sacrifice
- Blood samples collected from: retrobulbar venous plexus under isoflurane anesthesia
- Anesthesia: yes, isoflurane

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: viability (check for dead or moribund animals), behavioral changes, reaction to treatment, illness and the signs of abortion or premature delivery, appearance, change and disappearance of clinical signs

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT AND BODY WEIGHT GAIN: Yes
- Time schedule for body weight examinations: first measurement on Day 0 of pregnancy, daily afterwards
- Time schedule for body weight gain examination: calculated in intervals (gestation day 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20)

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

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily inspection of drinking water bottles

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: thyroid and gravid uterus including cervix were weighed, macroscopic examination of the internal organs of the dams were performed

OTHER: A macroscopic examination of the internal organs of the dams was performed

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, incl. distributions in the uterine horns
- Number of implantations: Yes, incl. distributions in the uterine horns
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- No. of animals: 20 for the control, 100, and 300 mg/kg bw/day groups, 19 for the 1000/750 mg/kg bw/day group
- Plasma: No
- Serum: Yes
- Volume collected
: 900 µL (300 µL each for T3, T4 and TSH analysis)

Fetal examinations:

- No. of fetuses examined: 20 litters/group examined
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Yes
- Parameters examined:
- Macroscopic inspection (gross evaluation)
- Number of fetuses (alive and dead) and placentae (location in the uterus and assignment of fetuses)
- Sex and viability
- Number and size of resorptions
- Corpora lutea in the ovaries
- Implantations and location of fetuses in the uterus
- Weights of fetuses and weights of the placentae
- Ano-genital distance (AGD) of live fetuses
- External inspection for damages (especially malformations)
- Determination of number and kind of retardations, variations or malformations
- Comparison of external sex with internal (gonadal) sex
- Examination of incomplete testicular descent/cryptorchism in male fetuses

Statistics:

Parametrical data:
Homogeneity of variances and normality of distribution were tested using the BARTLETT's and SHAPIRO-WILK's test. In case of heterogeneity and/or non-normalityof distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Non-parametrical data:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01)
or
Chi2 test, n ≥ 100 (p ≤ 0.05 and p ≤ 0.01)
The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy or findings during the external or internal macroscopic examination of the fetuses) or an internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).

Indices:

Malformed fetuses:
Total malformation rate [%] = (malformed fetuses per group / fetuses per group) x 100

Fetuses with variations:
Total variation rate [%] = (fetuses per group with variations / fetuses per group) x 100

Fetuses with retardations:
Total retardation rate [%] = (fetuses per group with retardations / fetuses per group) x 100

Group indices:
Pre-implantation loss [%] = [(corpora lutea per group - implantations per group) / corpora lutea per group] x 100

Post-implantation loss [%] = [(implantations per group - living fetuses per group) / implantations per group] x 100

Indices per litter:
Pre-implantation loss [%] = sum of pre-implantation losses per litter in a group [%] / number of litters in a group

Post-implantation loss [%] = sum of post-implantation losses per litter in a group [%] / number of litters in a group

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

1000/750 mg/kg bw/day: Salivation (21/21), decreased water consumption at the beginning of dosing (4/21), increased water consumption during second half of dosing period (20/21), breathing sounds (6/21), clear discharge from vagina and anus (5/21), reduced motility (4/21), hemorrhagic nose/snout (4/21), soft feces (4/21) and piloerection (3/21); adverse.
100 and 300 mg/kg bw/day: Salivation (3/20 at 100 mg/kg bw/day, 11/20 at 300 mg/kg bw/day) only for short period; non-adverse.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

1000/750 mg/kg bw/day: One animal prematurely sacrificed on GD 8 due to severe clinical signs (gasping, severe salivation and a hemorrhagic nose/snout).
100 mg/kg bw/day: One animal found dead on GD 11 due to misgavage.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000/750 mg/kg bw/day: Decreased body weight from GD 8 - GD 21 (at maximum 12.3% below the value of controls), reduced body weight gain (GD 0 - GD 20 and GD 6 - GD 20); adverse.
300 mg/kg bw/day: Decreased body weight gain (GD 6 - GD 20); non-adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1000/750 mg/kg bw/day: Decreased food consumption from GD 6 - GD 16 (at maximum 40.9% below the value of the controls); adverse.

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

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

1000/750 mg/kg bw/day: Statistically significant decreased gravid uterus weight (22.0% below the value of the controls)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

300 and 1000/750 mg/kg bw/day: Thickened and yellow discoloured cardia region of the stomach (3/20 at 300 mg/kg bw/day, 9/21 at 1000/750 mg/kg bw/day), hemorrhagic foci (2/20 at 300 mg/kg bw/day).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In all groups thyroids revealed single or multiple keratinized cysts; incidental.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone levels
300 and 1000/750 mg/kg bw/day: Statistically significant, dose-response related decrease of T3 and T4 serum levels (T3: 30.2% or 29.8% below controls; T4: 19.8% or 28.7% below controls) and dose-dependent increase of TSH serum levels (not statistically significant); hormone levels were within the range of historical control data of the test facility and no correlation with thyroid weights and histopathology noted.

Details on results:

MORTALITY
In the high dose group (1000 mg/kg bw/day) one dam was prematurely sacrificed on GD 8 due to signs of severe toxicity (gasping, severe salivation and a hemorrhagic nose/snout). In addition at necropsy, this dam had an emaciated body and highly dilated and inflated intestines without content. Evaluation of the pregnancy status by staining of the uterus according to SALEWSKI revealed that the animal was not pregnant. As a consequence and due to further signs of toxicity (clinical signs, reduced body weight, reduced food consumption), the high dose level was reduced from 1000 mg/kg bw/day to 750 mg/kg bw/day thereafter. In the low dose group (100 mg/kg bw/day), one animal was found dead on GD 11. Necropsy revealed a thorax filled with liquid and dark-red discolored lungs. Therefore, the premature death of this animal was considered to be due to a misgavage and the dam was excluded from the study. Therefore, no premature test item-related deaths occurred in the control group and the low- and mid-dose group (100 or 300 mg/kg bw/day).

CLINICAL SIGNS
In the high-dose group (1000/750 mg/kg bw/day), several clinical signs and changes of the external appearance and the feces were noted. Salivation and increased water consumption were noted for almost all animals of the high-dose group (1000/750 mg/kg bw/day) and therefore, were considered to be test item-related. Breathing sounds, clear discharge from vagina and anus, decreased water consumption, reduced motility, hemorrhagic nose/snout, soft feces and piloerection were considered to be test item-related and adverse.
No test item-related clinical signs or changes in the external appearance that were considered to be of toxicological relevance were noted in the control group and the low- and mid-dose groups (100 or 300 mg/kg bw/day). Salivation was noted for 3/20 dams of the low dose group (100 mg/kg bw/day) and 11/20 animals of the intermediate dose group (300 mg/kg bw/day). However, as salivation was noted for only up to 5 days per animal, salivation was considered to be due to high amounts of test item administered and/or local adverse effects in the stomach and therefore, not toxicologically relevant. Furthermore, breathing sounds were noted for one mid-dose dam on GD 17.

BODY WEIGHT DEVELOPMENT
In the high-dose group (1000/750 mg/kg bw/day), a decreased body weight was noted from GD 8 until study termination on GD 21 (at maximum 12.3% below the value of the control group on GD 20, statistically significant at p ≤ 0.01). This distinct decrease of the body weight that lasted for almost the complete dosing period, was considered to be toxicologically relevant. The decreased body weight in the high-dose group (1000/750 mg/kg bw/day) also led to a toxicologically relevant reduction for the body weight gain from GD 0 to GD 20 and from GD 6 to GD 20.
No test item-related differences in body weight were noted for the low- and mid-dose groups (100 and 300 mg/kg bw/day, respectively). In accordance with the body weight, also no test item-related difference between the control group and the animals treated with 100 or 300 mg/kg bw/day was noted for the body weight gain from GD 0 to 20 and in the dosing period from GD 6 to GD 20. In the mid-dose group (300 mg/kg bw/day), a statistically significantly lower body weight gain was noted between GD 6 and GD 20 (7.8% below the value of the control group). However, this small difference in body weight gain was considered to be not toxicologically relevant as it was < 10%.

FOOD AND DRINKING WATER CONSUMPTION
In the high-dose group (1000/750 mg/kg bw/day), a reduction of the food consumption was noted from GD 6 to GD 16 (at maximum 40.9% below the value of the control group between gestation days 7 to 8, with the exception of the period from GD 13 to GD 14). The long-lasting and distinct reduction in food consumption was considered to be test item-related. Almost all high dose females (1000/750 mg/kg bw/day) displayed increased water consumption and 4 females had a decreased water consumption. The females nos. 76 and 77 had a decreased water consumption on GD 18 and 19 or on GD 19 and increased water consumption from GD 20 until study termination on GD 21. Female no. 95 displayed an increased water consumption on GD 13 and a decreased water consumption from GD 14 to GD 21. Animal no. 78 was noted with decreased water consumption only from GD 18 to GD 21. The other animals displayed increased water consumption only.
At the start of the dosing period between GD 6 and GD 9, a reduced food consumption was noted for the low- (100 mg/kg bw/day) and mid- (300 mg/kg bw/day) dose groups (at maximum 9.9% below the value of the control group between GD 8 and GD 9 for the mid-dose group). However, these reductions in the food consumption for the low- and mid-dose groups were considered to be spontaneous. In addition, single occurrences of a statistically significantly reduced food consumption from GD 10 to GD 11 and from GD 15 to GD 16 for the mid-dose group (7.7% or 6.9% below the value of the control group) were considered to be spontaneous.

THYROID HORMONE LEVELS
A statistically significant dose-response related decrease was noted for the serum levels of T3 and T4 for the animals dosed with 300 or 1000/750 mg/kg bw/day (T3: 30.2% or 29.8% below the value of the control group; T4: 19.8% or 28.7% below the value of the control group). The serum levels of TSH were in tendency dose-dependently increased (not statistically significant). However, the levels of the mid- (300 mg/kg bw/day) and high-dose group (1000/750 mg/kg bw/day) for T3, T4 and TSH were within the range of the LPT historical control data and no correlation to thyroid weights and histopathology was noted in this study and in the 90-day sub-chronic toxicity study (LPT Study No. 37647; LPT, 2020a in section 7.5 of the IUCLID dossier). Therefore, the decreased levels of T3 and T4 and the increased levels of TSH observed in the mid- and high-dose groups are considered to be not toxicologically relevant.

NECROPSY
No test item-related observations were noted for the dams of the low-dose group (100 mg/kg bw/day) during the macroscopic inspection of the organs and tissues. In the mid-dose group (300 mg/kg bw/day), in 3/20 dams the cardia region of the stomach was thickened, yellow discolored and in two cases also with hemorrhagic foci. In the high-dose group (1000/750 mg/kg bw/day), thickened and discolored cardia region of the stomach was noted for 9/21 animals (20 dams and animal no. 86 with a total loss of implantations). Due to the dose-dependence relationship, the local changes in form of the thickened cardia region were considered to be test item-related and are believed to be caused by the chemical (irritating/corrosive) properties of the test item.

HISTOPATHOLOGY AND THYROID WEIGHTS
In all groups, histopathological examination of the thyroids revealed single or multiple keratinised cysts. However, these findings were considered to be coincidental and not test item-related. No test item-related differences to the control group were noted for the absolute thyroid weights of the animals in the dose groups (100, 300 or 1000/750 mg/kg bw/day).

GRAVID UTERUS WEIGHT, CARCASS WEIGHT AND BODY WEIGHT GAIN FROM GD 6
In the high-dose group (1000/750 mg/kg bw/day), statistically significant reductions were noted for the gravid uterus weight (22.0% below the value of the control group) and for the carcass weight (8.2% below the value of the control group). No test item-related differences were noted between the gravid uterus and carcass weight of the control dams and the dams of the low- and mid-dose groups (100 and 300 mg/kg bw/day, respectively).
In the high-dose group (1000/750 mg/kg bw/day), a decrease was noted also for the net body weight gain between GD 6 and GD 21 (-18.3 g in the high dose group compared to +3.2 g in the control group). The distinctly reduced net body weight gain was considered to be test item-related. For the net body weight from GD 6 to 21, no test item-related differences were noted between the dams of the control group and the dams of the low- and mid-dose groups (100 an 300 mg/kg bw/day).

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

1000/750 mg/kg bw/day: Due to an increased number of resorptions, the index of post-implantation loss is also increased.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

1000/750 mg/kg bw/day: Increased number of resorptions (33 resorptions vs. 13 resorptions in the control group).

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

In the high-dose group (1000/750 mg/kg bw/day), an increase was noted for the number of resorptions. In detail, in the control group 13 resorptions were noted and 14 resorptions each occurred in the low- and mid-dose groups. In the high-dose group 33 resorptions were noted, including animal no. 86 that lost all of its 15 implantations. Furthermore, a statistically significantly increased number of late resorptions was noted for the high-dose group (1000/750 mg/kg bw/day; 4 dams with one late resorption each in the high-dose group compared to no late resorptions in the control group). Since also the number of late resorption was above the upper limit of the LPT historical control data range, the increased number of resorptions noted for the high dose group was considered to be test item-related.
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the low- and mid-dose groups (100 and 300 mg/kg bw/day, respectively).

Key result

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

early or late resorptions

Key result

Dose descriptor:

NOAEL

Remarks:

local toxicity

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

gross pathology

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: local changes in cardia region of stomach

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000/750 mg/kg bw/day: Decreased weights of male and female fetuses, alone and for male and female fetuses combined (12.8%, 13.5% and 12.9% below the value of the controls), decreased fetal weights were considered secondary to reduced body weight (gain) and food consumption of dams.

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

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

100 mg/kg bw/day: One fetus with open cranial roof (cranioschisis) and one fetus with a short tail.

Skeletal malformations:

no effects observed

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

100 mg/kg bw/day: One open skullcap and one case of brachycaudia (refer also to cranioschisis and short tail reported under 'External malformations').

Other effects:

no effects observed

Description (incidence and severity):

Testicular development
No cryptorchism and no testicular malposition noted.

Details on embryotoxic / teratogenic effects:

No dead fetus was noted and no test item-related differences between the ratio of male and female fetuses were noted between the control group and the dose groups (100, 300 or 1000/750 mg/kg bw/day).
The placental weights showed no test item-related differences between the control group and the dose groups and no test item-related differences were noted for the fetal weights of the low and intermediate dose group (100 and 300 mg/kg bw/day) compared to the control group. For the fetal weights of the high dose group (1000/750 mg/kg bw/day), however, a decrease was noted for the male and female fetuses alone and also for the male and female fetuses combined (12.8%, 13.5% and 12.9% below the value of the control group, statistically significant). The decreased fetal weights were considered to be secondary to the reduced body weight (gain) and the reduced food consumption of the dams as the low fetus weights of the individual animals correspond to a very low food consumption and reduced net body weight gain of the dams.
No test item-related difference in the number of runts were noted for dose groups (100, 300 and 1000/750 mg/kg bw/day) in comparison to the control group. One runt each was noted in the control group and the mid-dose group (300 mg/kg bw/day), three runts in the low-dose group (100 mg/kg bw/day) and two runts were noted in the high-dose group (1000/750 mg/kg bw/day).
No test item-related differences to the control group were noted for the fetal anogenital distance of the dose groups (100, 300 and 1000/750 mg/kg bw/day).

MACROSCOPIC INSPECTION OF FETUSES
No macroscopically visible external observations were noted for the fetuses of the control group and the fetuses of the mid- and high-dose groups (300 and 1000/750 mg/kg bw/day) during the external inspection at laparotomy. In the low-dose group (100 mg/kg bw/day), two fetuses with a malformation were noted: One fetus was noted with an open cranial roof (cranioschisis) and one fetus had a short tail. The observations were also noted during the soft tissue examination of these two fetuses. However, as no malformations were noted in the mid- (300 mg/kg bw/day) and high-dose groups (1000/750 mg/kg bw/day), the occurrence of two fetuses with malformations in the low-dose group was considered to be spontaneous and not test item-related.
The macroscopic inspection of the organs and tissues for gross alterations at laparotomy revealed no malformations or variations for the fetuses of the control group and the fetuses of the dose groups (100, 300 and 1000/750 mg/kg bw/day).
No cryptorchism and no testicular malposition were noted during assessment of the testicular development of the male fetuses of the control group and the dose groups (100, 300 and 1000/750 mg/kg bw/day).

SKELETAL EXAMINATION
Malformations:
No skeletal malformations were noted for the fetuses of the control group and the test item-treated groups (100, 300 and 1000/750 mg/kg bw/day) during the skeletal examination according to DAWSON.

Variations:
Skeletal variations were noted for the ribs (less than 13 ribs ossified or ribs wavy) and the sternum (bipartite, dumbbell-shaped or misaligned to a slight degree). No test item-related difference in the incidence of the observed skeletal variations in comparison to the control group was noted for the fetuses of the treatment groups (100, 300 and 1000/750 mg/kg bw/day).

Retardations:
Retardations (i.e. delayed ossifications) were related to the skull (incomplete ossification of frontal, parietal, interparietal and/or supraoccipital areas), the hyoid (unossified), the sternum (sternebra(e) incompletely ossified, reduced in size or unossified), the thoracic vertebral bodies (bipartite or dumbbell-shaped), the caudal vertebral bodies (only one body ossified or all bodies unossified), the sacral vertebral bodies (unossified), the os pubis (incompletely ossified) and the metacarpalia/metatarsalia (absence of ossification in metacarpalia/metatarsalia 2 to 5). No test item-related difference in the incidence of skeletal retardations at 100, 300 and 1000/750 mg/kg bw/day was noted during skeletal examination according to DAWSON.

SOFT TISSUE EXAMINATION
Malformations
No malformations were noted for the fetuses of the control group and the fetuses of the mid- and high-dose groups (300 and 1000/750 mg/kg bw/day) during the soft tissue examination according to WILSON. In the low-dose group (100 mg/kg bw/day), two malformations were noted in form of an open skullcap and in form of a brachycaudia (see also the observations of cranioschisis and short tail). However, as no malformations were noted in the mid- and high-dose groups, the occurrence of two fetuses with malformations in the low-dose group was considered to be spontaneous and not test item-related.

Variations
During the examination of the organs and tissues according to WILSON, variations were noted for the brain (dilatation of the 4th cerebral ventricle or distinct cystic area in the cerebrum), the kidneys (uni- or bilateral dilatation of the renal pelvis or malpositioned) and the liver (hemorrhagic focus/foci). No test item-related and statistically significant differences in the incidences of the observed variations were noted between the control group and the treatment groups (100, 300 and 1000/750 mg/kg bw/day).

Unclassified observations
No unclassified observations were noted for the control group. An unclassified observation in form of a thoracic cavity filled with blood was noted for one fetus each of the low- and mid-dose groups (100 and 300 mg/kg bw/day, respectively) and for three fetuses of the high-dose group (1000/750 mg/kg bw/day). This observation was considered to be a preparation-induced artefact.

ASSESSMENT OF FETAL ALTERATIONS
No test item-related malformations or variations were noted during the macroscopic inspection at laparotomy (including an external inspection and a gross inspection of the organs), the skeletal examination according to DAWSON and the soft tissue examination according to WILSON). Furthermore, no test item-related retardations (delay in ossification) were noted in any of the dose groups (100, 300 and 1000/750 mg/kg bw/day).

Key result

Dose descriptor:

NOAEL

Remarks:

development

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: cranium

external: tail

Description (incidence and severity):

100 mg/kg bw/day: one fetus with open cranial roof (cranioschisis) and one fetus had a short tail, observations were considered spontaneous and not treatment-related.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

no

Detailed tables summarising study data are provided under 'Attached background material'. Summary tables demonstrating key results are provided below.

 

Table 1: Summary of animals examined

	Group 1 Control	Group 2 100 mg/kg bw/day	Group 3 300 mg/kg bw/day	Group 4 1000/750 mg/kg bw/day
Treated dams	25	25	25	25
Not pregnant dams	2	0	1	0
Dams with non-test item-related premature death	0	1*	0	0
Prematurely sacrificed dams	0	0	0	1**
Dams without viable fetuses	0	0	0	1***
Not evaluated dams (spare animals)	3	4	4	3
Evaluated litters	20	20	20	20

*: The death of dam no. 26 that was found dead on GD 11 was considered to be due to a misgavage. Therefore, dam no. 26 was excluded from the study.

**: Dam no. 82 was prematurely sacrificed due to severe signs of toxicity.

***: Dam no. 86 was noted with resorptions of all implants.

 

Table 2: Summary of animals evaluated

	Group 1 Control	Group 2 100 mg/kg bw/day	Group 3 300 mg/kg bw/day	Group 4 1000/750 mg/kg bw/day
Animal nos. of mated rats	1 - 25	26 - 50	51- 75	76 - 100
Animal nos. with evaluable litters at laparotomy	1, 4 - 22	27 - 46	51, 53 - 71	76 - 81, 83 - 85, 87 - 97
Dams not pregnant (animal nos.)	2, 3	None	52	None
Dams with non-test itemrelated premature death (animals nos.)	None	26*	None	None
Prematurely sacrificed dams (animal nos.)	None	None	None	82**
Dams without viable fetuses (animal nos.)	None	None	None	86***
Spare animals (animal nos.)	23 - 25	47 - 50	72 - 75	98 - 100

*: The death of dam no. 26 that was found dead on GD 11 was considered to be due to a misgavage. Therefore, dam no. 26 was excluded from the study.

**: Dam no. 82 was prematurely sacrificed due to severe signs of toxicity.

***: Dam no. 86 was noted with resorption of all implants.

 

Table 3: Clinical signs, changes of external appearance and the feces noted for the high dose group

	Observations in group 4 (1000/750 mg test item/kg bw/day)*
Observation	Affected dams	First to last day seen	Number of days observed (min - max) in at least 1 animal
Salivation (slight to severe)	21/21	GD 6 - GD 21	1 - 11
Increased water consumption	20/21	GD 11 - GD 21	1 - 9
Breathing sounds	6/21	GD 7 - GD 21	2 - 5
Discharge from vagina and anus (clear)	5/21	GD 7 - GD 16	1 - 6
Decreased water consumption	4/21	GD 14 - GD 21	1 - 8
Motility reduced	4/21	GD 9 - GD 19	1 - 4
Hemorrhagic nose/snout	4/21	GD 8 - GD 18	1
Soft feces	4/21	GD 11 - GD 16	2 - 5
Piloerection	3/21	GD 10 - GD 21	1 - 12

*: The prematurely sacrificed animal no. 82 was excluded in this table.

 

Table 4: Body weight gain from GD0 to GD20 and from GD6 to GD20

Group	Time interval Gestation day 0 - 20 (whole study period)			Time interval Gestation day 6 - 20 (whole treatment period)
	Gain [g]	Gain [%]	Difference to control [%]	Gain [g]	Gain [%]	Difference to control [%]
Control	+171.8	+71.0	n.a.	+131.6	+46.6	n.a.
Group 2 100 mg/kg bw/day	+163.3	+67.9	-5.0	+124.5	+44.5	-5.4
Group 3 300 mg/kg bw/day	+164.8	+68.6	-4.1	+121.3*	+42.7	-7.8
Group 4 1000/750 mg/kg bw/day	+120.6**	+49.8	-29.8	+82.7**	+29.4	-37.1

n.a.: Not applicable

*/**: Statistically significant at p ≤ 0.05/0.01 (DUNNETT test).

 

Table 5: Thyroid hormone levels determined in this study and LPT background data on T3 and T4 levels

Thyroid hormone levels	Values observed in this study [mean per dam]	LPT Background Data*** Mean value ± SD (range) of the individual control groups (n = 5 control groups; data taken from 2019 to June 2020)
T3 [ng/mL]	Control: 2.46 Group 2: 2.21 Group 3: 1.72 **/**** Group 4: 1.72 **/****	2.17 ± 0.86 1.22 - 8.64	control groups
T4 [nmol/L]	Control: 19.14 Group 2: 18.47 Group 3: 15.35 */**** Group 4: 13.66 **/****	20.27 ± 4.71 11.82 - 37.82	control groups
TSH (ng/mL)	Control: 1.03 Group 2: 1.07 Group 3: 1.49 Group 4: 2.28	0.85 ± 0.87 0.08 - 4.75	control groups

*/**: Statistically significant at p ≤ 0.05/0.01 (DUNNETT test).

***: Not audited by QAU.

****: Considered to be spontaneous as incidences were within LPT background data range

 

Table 6: Macroscopic changes noted during necropsy

Group	Animal no.	Observation
Group 1 Control	-	No observation noted
Group 2 (100 mg/kg bw/day)	No. 26 (PD)	Thorax: filled with liquid Lungs: dark-red discolored
	No. 35	Uterus: amniotic fluid red discolored
Group 3 (300 mg/kg bw/day)	No. 54	Stomach: cardia region thickened, yellow discolored
	No. 55	Stomach: cardia thickened, yellow discoloured, 2 hemorrhagic foci (approx. 1 mm in diameter)
	No. 59	Stomach: cardia region thickened, yellow discoloured, 2 hemorrhagic foci (approx. 1-2 mm in diameter)
Group 4 (1000/750 mg/kg bw/day)	No. 78	Intestines: inflated Stomach: cardia region thickened, white discolored
	No. 80	Stomach: cardia region thickened, yellow discolored
	No. 82 (PS, NP)	Body: emaciated Intestines: without content, strongly dilated, inflated
	No. 85	Stomach: cardia region thickened, yellowish discolored
	No. 87	Stomach: mucosa thickened, cardia region yellowish discolored
	No. 88	Stomach: mucosa thickened in cardia region
	No. 89	Stomach: cardia region thickened, yellowish discolored
	No. 90	Stomach: cardia region thickened, yellowish discolored
	No. 91	Stomach: cardia region thickened, white discolored
	No. 92	Stomach: cardia region thickened, yellowish discolored

PD: Premature death

PS: Premature sacrifice

NP: Not pregnant

 

Table 7: Overview of reproduction parameters

Parameter		Group 1 Control (n = 20)	Group 2 (100 mg/kg bw/day) (n = 20)	Group 3 (300 mg/kg bw/day) (n = 20)	Group 4 (1000/750 mg/kg bw/day) (n = 21)
Corpora lutea	total mean per dam	336 16.8	316 15.8	321 16.1	337 16.0
Implantation sites	total mean per dam	330 16.5	316 15.8	319 16.0	330 15.7
Resorptions	total mean per dam	13 0.7	14 0.7	14 0.7	33 1.6
Early resorptions	total mean per dam	13 0.7	14 0.7	14 0.7	29 1.4
Late resorptions	total mean per dam	0 0.0	0 0.0	0 0.0	4 0.2*
Live fetuses	total mean per dam	317 15.9	303 15.2	305 15.3	297 14.9
Dead fetuses	total	0	0	0	0
Pre-implantation loss [%]	per group mean per dam	1.8 1.7	0.0 0.0	0.6 0.6	2.1 2.0
Post-implantation loss [%]	per group mean per dam	3.9 3.8	4.4 4.5	4.4 4.2	10.0 10.2

*: Statistically significant at p ≤ 0.05 (DUNNETT test).

Statistical analyses were performed for the mean values per dam using an ANOVA/DUNNETT test.

 

Table 8: LPT background data on number of late resorptions

Reproduction parameters	Values observed in this study [mean per dam]	LPT Background DataJ** Mean value ± SD (range) of the individual control or test groups (n = 12 control or data taken from 2016 to July 2017)
Late resorptions	Control: 0.0 Group 2: 0.0 Group 3: 0.0 Group 4: 0.2*	0.03 ± 0.05 0.0 - 0.1	control groups

*: Statistically significant at p ≤ 0.05 (DUNNETT test).

**: Not audited by QAU

 

Table 9: Male to female ratio for the fetuses of the test groups

	Ratio [male/female fetuses]
Control	1.10
100 mg/kg bw/day	1.06
300 mg/kg bw/day	0.97
1000/750 mg/kg bw/day	1.14

 

Table 10: Statistically significant, considered to be not test item-related differences in the incidence of fetal skeletal retardations

Skeletal retardations	Values observed in this study [fetal incidence in % per group]	LPT Background Data*** Mean value ± SD (range) of the individual control or test groups [fetal incidence in % per group] (n = 15 control or n = 45 test item groups, data taken from 2016 to July 2017)
Absence of ossification in metacarpalia 2 to 5	Control: 0.6 Group 2: 0.7 Group 3: 0.7 Group 4: 7.4 **/****	3.6 ± 4.0 0.0 - 15.0	control groups
Absence of ossification in metatarsalia 2 to 5	Group 2: 0.0 Group 3: 0.0 Group 4: 2.7 */****	1.2 ± 1.8 0.0 - 4.6	control groups
Caudal vertebral bodies only one body ossified	Control: 0.0 Group 2: 0.0 Group 3: 0.0 Group 4: 2.7 */****	1.0 ± 2.1 0.0 - 7.9	control groups
Hyoid unossified	Control: 26.4 Group 2: 30.9 Group 3: 25.5 Group 4: 12.2 **/*****	54.2 ± 16.3 24.3 - 77.6	control groups
Sternebra(e) incompletely ossified	Control: 0.0 Group 2: 0.0 Group 3: 2.0 Group 4: 2.7 */****	10.3 ± 6.1 3.4 - 26.7	control groups
Thoracic vertebral body/bodies bipartite	Control: 5.0 Group 2: 0.7 */***** Group 3: 0.7 */***** Group 4: 1.4 */*****	2.6 ± 1.5 0.7 - 5.1	control groups

*/**: (p ≤ 0.05 / p ≤ 0.01) Fisher or Chi² - test

***: Not audited by QAU.

****: Considered to be spontaneous as incidences were within LPT background data range.

*****: A decreased incidence was considered to be spontaneous.

 

Table 11: Results of the test item formulation analysis

Parameter	Sampling	Range of % nominal concentration
Homogeneity	at the start of administration, during administration and before administration to the last animal of each dose group	93.8% - 105.9%*
Concentration	before administration of the last animal of each dose group at a time when the majority of animals was dosed	95.0% - 103.5%

*: Contains results before and after high dose level adjustment from 1000 mg/kg to 750 mg/kg.

 

Conclusions:

The pre-natal developmental toxicity in rats after oral administration by gavage was investigated according to OECD guideline 414 under GLP conditions. N-methyl-N-[C18-(unsaturated)alkanoyl]glycine exhibited distinct maternal toxicity at doses > 300 mg/kg bw/day. Reduced pup weights observed were a secondary reaction to maternal toxicity. The No-Observed-Adverse-Effect-Levels (NOAEL) for maternal systemic, maternal developmental and fetal developmental toxicity were, therefore, established to be 300 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to analogue justification report provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

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.

Remarks on result:

other: Source: CAS 137-16-6, Envigo, 2017

Key result

Abnormalities:

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.

Remarks on result:

other: Source: CAS 137-16-6, Envigo, 2017

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. Source: CAS 137-16-6, Envigo, 2017

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.

Executive summary:

As explained in the category/analogue justification, the differences in molecular structure between the target and the source substances are unlikely to lead to differences with respect to toxicity to reproduction.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises adequate and reliable (Klimisch score 1) studies conducted with the registered substance and from a source substance with similar structures and intrinsic properties. Read-across is justified based on common precursors and metabolic degradation products and a consistent trend in toxicological profile. The selected studies are thus sufficient to fulfil the standard information requirements set out in Annexes VIII - X, Section 8.7, in accordance with Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH).

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Pre-natal developmental toxicity in a first species (rats)

The pre-natal developmental toxicity of N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC No. 701-177-3) was investigated in rats in a study according to OECD guideline 414 under GLP conditions (LPT, 2020b). The substance was administered orally (daily, 7 days/week) to groups of 25 pregnant female Sprague-Dawly (Crl:CD(SD)) rats at dose levels of 100, 300 and 1000/750 mg/kg bw/day from the 6th to 20th day of pregnancy. A similarly constituted control group received the vehicle (0.5% aqueous hydroxypropylmethylcellulose gel) only. Clinical signs, body weight development, food and water consumption were monitored during the duration of the study. At sacrifice on gestation day (GD) 21 all females were subjected to gross necropsy including examination of the uterine contents, gravid uterus weight, number of corpora lutea (incl. distributions in the uterine horns, number, position and type of implantation, foetal weight, sex, ano-genital distance, and external and internal macroscopic appearance. In addition, thyroid and thyroid hormones (T3, T4, TSH) were also evaluated for the dams. Half of each litter was examined for detailed skeletal development and the remaining half was subjected to detailed visceral examination.

No premature test item-related deaths occurred in the control group and the low- and mid-dose groups (100 and 300 mg/kg bw/day, respectively). In the high-dose group (1000 mg/kg bw/day) one dam was prematurely sacrificed on GD 8 due to signs of severe toxicity (gasping, severe salivation and a hemorrhagic nose/snout). As a consequence and due to further signs of toxicity (clinical signs, reduced body weight, reduced food consumption), the high-dose level was reduced from 1000 mg/kg bw/day to 750 mg/kg bw/day thereafter. In the low-dose group (100 mg/kg bw/day), one animal was found dead on GD 11. Necropsy revealed a thorax filled with liquid and dark-red discolored lungs. Therefore, the premature death of this animal was considered to be due to a misgavage and the dam was excluded from the study. In the high-dose group, slight to severe salivation, increased water consumption and further clinical signs and changes in the feces were noted. Test item-related reductions were noted in the food consumption of the high-dose group. Furthermore, a decrease in the body weight, the body weight gain and the carcass weight of the dams of the high-dose group was noted. Test substance-related local changes at necropsy were noted for the animals of the mid- and high-dose groups: a thickening and yellow or white discolouration of the cardia region of the stomach. No differences for the thyroid weights were noted and the histopathological examination of the thyroids did not reveal any test item-related changes. No toxicologically relevant differences were noted for the serum levels of T3, T4 and TSH. In the high-dose group, an increased number of resorptions and a decreased body weight of the pups were noted. No deaths of fetuses and no test item-related malformations, variations or retardations were noted. Since adverse effects on the pups were observed only at doses exhibiting distinct maternal toxicity and no teratogenic effect was noted in any dose group, the increased number of resorptions and decreased body weights of the pups are considered secondary effects of the maternal toxicity.

Based on the local changes in the stomach of the mid and high dose animals, the No-Observed-Adverse-Effect-Level (NOAEL) for local toxicity for the dams was 100 mg/kg bw/day while the NOAELs for maternal systemic toxicity and maternal developmental toxicity were found to be 300 mg/kg bw/day. With regard to fetal developmental toxicity, the NOAEL for fetal development was 300 mg/kg bw/day based on reduced pup weight. No teratogenic effect was observed.

 

Pre-natal developmental toxicity in a second species (rabbit)

No study investigating the pre-natal developmental toxicity of N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC No. 701-177-3) is available. Due to the high structural similarity to the analogue source substance sodium [dodecanoyl(methyl)amino]acetate (CAS No. 137-16-6), the pre-natal developmental toxicity and the lack of any teratogenic effect of the target substance in rabbits is predicted based on the study performed with the source substance.

 

Three groups of 22 females received the test item sodium N-lauroylsarcosinate (CAS No. 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 detailed internal 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/day. In addition, there was no clear effect of treatment on the body weight or food consumption performance of females receiving the test substance at 125 or 250 mg/kg bw/day when compared with that of the control group. The mean body weight loss of females exposed to 500 mg/kg bw/day 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/day 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/day. 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/day there was a slightly higher incidence of the minor fetal abnormality of 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/day, 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 toxicity and embryo-fetal development was concluded to be 500 mg/kg bw/day, corresponding to the highest dose tested.

 

Conclusion

Local toxicity due to the irritating/corrosive effect of the registered (target) substance N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC No. 701-177-3) in the stomach of the test animals was observed. As a consequence, severe maternal toxicity occurred, leading to reduced pup weights as a secondary effect. There are no indications of developmental toxicity other than secondary effects or any teratogenic effect of the target substance. No biologically relevant effects were detected in the study conducted with the source substance sodium [dodecanoyl(methyl)amino]acetate (CAS No. 137-16-6) in rabbits. Based on the reliable and adequate studies, no toxicologically relevant developmental or teratogenic effect is expected for the target substance with respect to pre-natal development of rabbits.

Justification for classification or non-classification

The available data on reproductive and developmental toxicity / teratogenicity with N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC No. 701-177-3) and the analogue source substance sodium [dodecanoyl(methyl)amino]acetate (CAS No. 137-16-6) do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification. An extended one-generation reproductive toxicity study according to OECD guideline 443 is proposed to meet the standard information requirements of Annex X, Section 8.7.3., of Regulation (EC) No. 1907/2006 (REACH). When study results are available, classification of N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC No. 701-177-3) will be re-assessed.