N,N'-ethane-1,2-diylbisoleamide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Waiving - Toxicity to reproduction - screening
Waiving - Toxicity to reproduction - extended one-generation reproduction toxicity study

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because a pre-natal developmental toxicity study is available

Reproductive effects observed:

not specified

Reference 2

Endpoint:

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

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

According to Regulation (EC) No 1907/2006, Annex VIII, column 2, a screening study for reproductive/developmental toxicity is not required as a pre-natal developmental toxicity study (Annex IX, 8.7.2) with a structurally related substance according to Annex XI, article 1.5 of the aforementioned regulation is available.

According to Regulation (EC) No 1907/2006, Annex IX, 8.7.3, column 1, an Extended One-Generation Reproductive Toxicity Study is not required as the available subchronic oral toxicity study conducted with the target substance and the chronic oral toxicity study conducted with a structurally related substance according to Annex XI, article 1.5 of the aforementioned regulation do not indicate adverse effects on reproductive organs or tissues. In addition, the available developmental toxicity studies with a structurally related substance did not reveal concerns in regard to reproductive toxicity. Therefore, on behalf of animal welfare, the conduct of animal studies on reproductive toxicity with the target substance by any route of exposure is considered scientifically unjustified.

Effects on developmental toxicity

Description of key information

Prenatal developmental toxicity (OECD 414), based on read-across from Amides, C16-C18 (even), N,N’-ethylenebis:
NOAEL maternal ≥ 1000 mg/kg bw/day
NOAEL developmental ≥ 1000 mg/kg bw/day 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

27 Nov - 24 Dec 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance N,N’-Ethylenebis(stearamide). According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy S.r.l., San Pietro al Natisone, Italy
- Age at study initiation: 9 weeks (female) and 11 weeks (male)
- Weight at study initiation: 191-221 g (female) and at least 316 g (male)
- Housing: before and after pairing, the animals were housed in groups of max. 5 animals per sex in clear polycarbonate cages (59 x 38.5 x 20 cm) with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray was equipped with absorbent paper which was inspected and changed at least 3 times per week. During the mating period, each one male and one female were housed in clear polycarbonate cages (43 x 27 x 18 cm) with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray was equipped with absorbent material which was inspected and changed daily.
- Diet: laboratory rodent diet (4 RF 21, Mucedola S.r.l., Settimo Milanese, Italy), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15-25
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 27 Nov 2008 To: 24 Dec 2008

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test substance was suspended in the vehicle. The formulatiuons were prepaired daily and concentrations were calculated and expressed in terms of the test item as supplied.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical verification of the test substance in corn oil was performed using Gas Chromatography with flame ionization detection (GC/FID). The stability of the test substance formulations was verified in the range of 10-100 mg/mL for a period of 24 h at room temperature. Samples of the formulations prepared during Weeks 1 and 3 of the study were analysed to check the homogeneity and concentration. All parameters were within the range limits of the analytical method.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1 in the home cage of the male
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy

Duration of treatment / exposure:

Day 6-19 post coitum

Frequency of treatment:

once a day

Duration of test:

Day 20 post coitum

No. of animals per sex per dose:

24 mated females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on preliminary study

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: throughout the study, all animals were checked early on each working day and again in the afternoon. At weekends and public holidays, a similar procedure was followed except that the final check was carried out at approximately mid-day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all clinical signs were recorded for individual animals from allocation to sacrifice.
Once daily before commencement of treatment and once daily at approximately 1-1.5 hours after treatment, each animal was observed and any clinical signs were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: all animals were weighed on Days 0, 3, 6, 9, 12, 15 and 20 post coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, on Days 3, 6, 9, 12, 15 and 20 post coitum starting from Day 0 post coitum.

POST-MORTEM EXAMINATIONS: Yes, the animals were killed with carbon dioxide on Day 20 post coitum and necropsied , all foetuses were sacrificed by hypothermia. Necropsy: the clinical history of the animal was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices) and changes were noted.
- Sacrifice on gestation Day 20

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter; number, sex and weight of all live foetuses; number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing); number of intra-uterine deaths classified as early resorptions (only placental remnants visible) and late resorptions (placental and foetal remnants visible).
- Soft tissue examinations: Yes: half per litter; fixed-visceral examination of all groups, gross evaluation of placentae.
- Skeletal examinations: Yes: half per litter; skeletal examinations were performed in all groups. Structural deviations were classified as follows: Malformations: major abnormalities that are rare and/or affect the survival or health of the species under investigation; Anomalies: minor abnormalities that are detected relatively frequently; Variants: changes that occur within the normal population under investigation and are unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that has otherwise followed a normal pattern of development.
- Head examinations: Yes, part of skeletal examination

Statistics:

Continuous variables: Dunnett's test or a modified t test
Non-continuous variables: Kruskal-Wallis test
Intergroup differences between the control and treated groups: non-parametric version of the Williams test

Indices:

Pre-implantation loss was calculated as a percentage from the formula: [(no. of corpora lutea - no. of implantations) x 100]/no. of corpora lutea
Post-implantation loss was calculated as a percentage from the formula: [(no. of implantation - no. of live young) x 100]/no. of implantations
Total implantation loss was calculated as a percentage from the formula: [(no. of corpora lutea - no. of live young) x 100]/no. of corpora lutea
Sex ratios of the foetuses were calculated as the percentage of males per litter.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical sign was detected in any animal during the whole study and no sign of reaction to treatment was noted.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In all treated females body weight and body weight gain were comparable to controls throughout the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No changes were detected in food consumption between treated and control females.

Food efficiency:

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant differences were noted in terminal body weight, uterus weight and absolute weight gain within the groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No findings were detected in treated females that could be considered treatment-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Unilateral implantation was detected in one low dose female.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

The number of females with live foetuses on gestation Day 20 was 24 each in the control, mid- and high dose groups and 23 in the low dose group.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

One female in the low dose group was found not pregnant at necropsy.

Other effects:

not specified

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
- No mortality occurred during the study.
- One female in the low dose group was found not pregnant at necropsy.
- Unilateral implantation was detected in one low dose female.
- The number of females with live foetuses on gestation Day 20 was 24 each in the control, mid- and high dose groups and 23 in the low dose group.
- No clinical sign was detected in any animal during the whole study and no sign of reaction to treatment was noted.
- In all treated females body weight and body weight gain were comparable to controls throughout the study.
- No changes were detected in food consumption between treated and control females.
- No significant differences were noted in terminal body weight, uterus weight and absolute weight gain within the groups.
- No findings were detected in treated females that could be considered treatment-related.

Key result

Dose descriptor:

NOAEL

Remarks:

maternal general toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed at this dose level.

Key result

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed at this dose level.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Two small foetuses (weight less than 2.7 g) were found in the control group and one in each of the treated groups.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Litter data and sex ratio were not affected by treatment.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter data and sex ratio were not affected by treatment.

Anogenital distance of all rodent fetuses:

not specified

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No abnormalities were detected at the external examination of all foetuses.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No changes that could be considered treatment-related were noted at skeletal examination of the foetuses between control and treated groups.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations were noted in three foetuses, one in the control group, one in the mid-dose group and one in the high dose group. These malformations and the other findings noted were considered to be incidental.

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Litter data and sex ratio were not affected by treatment.
- Two small foetuses (weight less than 2.7 g) were found in the control group and one in each of the treated groups.
- No other abnormalities were detected at the external examination of all foetuses.
- Malformations were noted in three foetuses, one in the control group, one in the mid-dose group and one in the high dose group. These malformations and the other findings noted were considered to be incidental.
- No changes that could be considered treatment-related were noted at skeletal examination of the foetuses between control and treated groups.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Neither clinical signs nor signs of reaction to treatment were noted in treated females. No significant differences were noted in body weight, food consumption, gravid uterus weight, litter data and macroscopic observation of treated females when compared to controls. No treatment-related changes were seen at the external, visceral and skeletal examination of foetuses from all groups.

Effect on developmental toxicity: 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 adequate, reliable (Klimisch score 2) and consistent studies from an analogue substance with similar structure and intrinsic properties. The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 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

Additional information

There are no data available on the toxicity to reproduction (development) of Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis. In order to fulfil the standard information requirements set out in Annex IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from a structurally related substance is conducted.

A detailed justification for the analogue approach and read-across is provided in Sections 7.1 and 13.

Reliable data is available from a prenatal developmental toxicity study of the structurally related substance Amides, C16-C18 (even), N,N'-ethylenebis, which was chosen as the key study, and a corresponding preliminary test which served as supporting information.

In the key study doses of 100, 300 and 1000 mg/kg bw/day, which had been analytically confirmed, were administered by oral gavage on gestational days 6 to 19 (Liberati, 2009). No maternal toxic effects were observed up to the highest dose of 1000 mg/kg bw/day. No mortality or clinical signs were observed during the study. Food consumption and body weight development were not affected by treatment with the test substance. The number of dams with live foetuses was comparable among all dose groups. Litter data and sex ratio were not affected by the treatment, and no visceral or skeletal abnormalities were noted within the foetuses which could be attributed to the test substance.

These results were supported by the corresponding range-finder study (Liberati, 2008), which was conducted with a reduced animal number, and no analysis of the administered dose. No mortality occurred during that study, either. All females were found pregnant at necropsy. No signs of toxicological significance were noted in any treated female. No signs of reaction to treatment were recorded at the daily post-dose observations performed 1 h after administration. Soft faeces on the cage tray were occasionally observed in the high dose group. Body weight and body weight gain in treated females were comparable to controls through the study. No differences were noted in litter data parameters between control and treated females. No relevant findings were reported at the macroscopic observation of females. Staining on different areas of the body surface noted in some animals was not considered treatment-related. In this range-finder study the foetuses were only examined externally, and no abnormalities were detected. Skeletal and visceral investigations did not detect any treatment-related malformations. For the embryo/fetotoxicity, the teratogenicity and the maternal toxicity a NOAEL of 1000 mg/kg bw/day was deduced.

Based on the results for Amides, C16-C18 (even), N,N'-ethylenebis, there is no developmental toxicity expected to occur for the structurally related Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis, either.

Justification for classification or non-classification

The available data on toxicity to reproduction of a substance structurally related to Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis, according to Regulation (EC) No 1907/2006, Annex XI, 1.5, do not meet the criteria for classification according to Regulation (EC) No 1272/2008; therefore, Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is not expected to exert a potential for reproduction toxicity, either, and the data are thus conclusive but not sufficient for classification.