Ethanol, 2,2'-iminobis-, N-(C16-18 and C18-unsatd. alkyl) derivs.

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19th June 2013 to 17 the December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test”

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology Studies, 12 NohSan No 8147 (24 November 2000)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-(octadec-9-enylimino)bisethanol

EC Number:

246-807-3

EC Name:

2,2'-(octadec-9-enylimino)bisethanol

Cas Number:

25307-17-9

Molecular formula:

C22H45NO2

IUPAC Name:

2,2'-(Octadec-9-enylimino)bisethanol

Test material form:

liquid

Details on test material:

- Chemical name : Bis(2-hydroxyethyl)oleylamine
- EC number : 246-807-3
- Purity: 100% UVCB
- Batch number: S-001018

Based on the qualitative and quantitative information on the composition, the sample used are representative of the boundary composition shared and agree by each registrant.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 190g to 269g.

The animals were housed individually in solid-floor polypropylene cages with stainless steel lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan UK, Oxon, UK) was used. Certificates of analysis of the batches of diet used are given in Appendix 14. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidity were included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; there were no deviations from these targets.

The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

As Arachis Oil was successfully used on both the twenty-eight day and ninety toxicity studies, the same vehicle and dosage (4 mL/kg body weight) was employed in this study. The stability and homogeneity of the test item formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services and showed the formulations to be stable for at least twenty one days at 4 °C. Formulations were therefore prepared in two separate bulk formulations (covering up to 9 days) and divided into daily aliquots and stored at approximately +4 °C in the dark.

Samples were taken of each test item formulation and were analyzed for concentration of 2,2'-(octadec-9-enylimino)bisethanol CAS No 25307-17-9 at Harlan Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within 94% to 104% of the nominal concentration and within acceptable limits of the nominal concentration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

Test Item
The test item described in the main part of this study was also used as the analytical standard.

Preparation of standard solutions
Stock solutins of test item in methanol were prepared for external standard calibration. An aliquot, 100 mg of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with methanol to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solutins were used to prepare working standard solutions in methanol with a concentration of 0.1 mg/mL.

Analysis of samples
The formulations recieved were extracted with methanol. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with methanol. This was then ultra-sonicated for 15 minutes and centrifuged to 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with methanol to achieve the working concentration.

Preparation of accuracy samples
Samples of Arachis Oil BP were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These sample were then prepared for analysis.

Instrumental Setip

HC system: Agilent Technologues 5890, incorporating autosampler and workstation
Column: DB-1 (15 m x 0.53 mm id x 1.5 micro-m film)
Oven temperature program: Oven: 200°C for 0 minute, with 10°C/minute to 300°C, for 12 minutes
Injection temperature: 300°C
Flame ionisation detector temperature: 300°C
Injection volume: 1 micro-litre
Retention time: ~ 4.5 mins

Details on mating procedure:

Not described in the study

Duration of treatment / exposure:

Between Days 5 and 19 of gestation, inclusive.

Frequency of treatment:

Daily

Duration of test:

20 days

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
15 mg/kg/day (3.75 mg/ml)
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50 mg/kg/day (12.5 mg/ml)
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
150 mg/kg/day (37.5 mg/ml)
Basis:
actual ingested

No. of animals per sex per dose:

24 females

Control animals:

yes, concurrent vehicle

Details on study design:

The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Justification
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Examinations

Maternal examinations:

Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. An additional observation was also performed five hours after dosing during the normal working week. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

Water Consumption
Water intake was observed daily by visual inspection of the water bottles for any overt changes.

Ovaries and uterine content:

Post Mortem
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:

i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight

Implantation types were divided into:

Early Death: No visible distinction between placental/decidual tissue and embryonic tissue

Late Death: Separate embryonic/fetal and placental tissue visible

Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes

All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):


Left Horn Cervix Right Horn

L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16

V = viable fetus

Fetal examinations:

The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.

Statistics:

The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:

Body weight and body weight change (including adjustment for the contribution of the gravid uterus), food consumption, gravid uterus weight, litter data and fetal litter and placental weights: Bartlett’s test for homogeneity of variance. Where the data were shown to be homogeneous one way analysis of variance and, if significant, Dunnett’s multiple comparison test was employed, where the data were found to non homogeneous Kruskal-Wallis and, if significant, pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test was employed. Fetal evaluation parameters, including skeletal or visceral findings were analyzed by Kruskal-Wallis and, if significant, Mann-Whitney ‘U’ test.

Probability values (p) are presented as follows:

p<0.01 **
p<0.05 *
p≥0.05 (not significant)

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No effects of any toxicological significance

Effect levels (maternal animals)

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter Responses
Litter Data and Litter Placental and Fetal Weights
There was no obvious effect of maternal treatment on the number of implantations, subsequent embryofetal survival and litter size, sex ratio and mean fetal, litter and placental weights on Day 20 of gestation at 15, 50 or 150 mg/kg bw/day.

At 150 mg/kg bw/day, mean pre-implantation loss was lower than control with differences attaining statistical significance. As animals were not dosed until implantation had occurred, these differences were incidental and unrelated to treatment.

At 15 and 50 mg/kg bw/day, higher mean female fetal weight and mean fetal weight attained statistical significance compared to control. In the absence of any similar increase in fetal weight at 150 mg/kg bw/day, this finding was considered to reflect normal biological variation and was unrelated to treatment.

Fetal Examination
Neither the type, incidence or distribution of findings observed externally at necropsy examination and subsequently during detailed visceral and skeletal assessment of the fetuses indicated any effect of treatment on fetal development.

Effect levels (fetuses)

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Mortality There were no unscheduled deaths during the study. Clinical Observations The low incidence of clinical sign observed during the study did not indicate any effect of treatment at 15, 50 or 150 mg/kg bw/day. Body Weight There was no effect of treatment on body weight or body weight gain, including when adjusted for the contribution of the gravid uterus, throughout the treatment period at 15, 50 or 150 mg/kg bw/day. Food Consumption There was no effect of treatment on food consumption throughout the treatment period at 15, 50 or 150 mg/kg bw/day. Water Consumption Daily visual inspection of water bottles did not reveal any overt intergroup differences. Post Mortem Studies No macroscopic abnormalities were detected for parental females at scheduled termination on Day 20 of gestation. Litter Responses Litter Data and Litter Placental and Fetal Weights There was no obvious effect of maternal treatment on the number of implantations, subsequent embryofetal survival and litter size, sex ratio and mean fetal, litter and placental weights on Day 20 of gestation at 15, 50 or 150 mg/kg bw/day. At 150 mg/kg bw/day, mean pre-implantation loss was lower than control with differences attaining statistical significance. As animals were not dosed until implantation had occurred, these differences were incidental and unrelated to treatment. At 15 and 50 mg/kg bw/day, higher mean female fetal weight and mean fetal weight attained statistical significance compared to control. In the absence of any similar increase in fetal weight at 150 mg/kg bw/day, this finding was considered to reflect normal biological variation and was unrelated to treatment. Fetal Examination Neither the type, incidence or distribution of findings observed externally at necropsy examination and subsequently during detailed visceral and skeletal assessment of the fetuses indicated any effect of treatment on fetal development.

Applicant's summary and conclusion

Conclusions:

The No Observed Effect Level (NOEL) for the pregnant females and the survival, growth and embryofetal development of the offspring was considered to be 150 mg/kg bw/day.

Executive summary:

Introduction

The study was designed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

 

The study was designed to comply with the following guidelines:

 ·        US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

·        Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

·        OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

·        Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

  

Methods

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD^®(SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 15, 50, and 150 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil) over the same treatment period to serve as a control. Clinical signs, body weight change, food and water consumptions were monitored during the study. 

 

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

 

Results…….

Adult Responses

Mortality: There were no unscheduled deaths during the study.

Clinical Observations: Clinical sign did not indicate any effect of treatment at 15, 50 or 150 mg/kg bw/day.

Body Weight: Body weight and body weight gain, including adjustment for the contribution of the gravid uterus, was unaffected by treatment at 15, 50 or 150 mg/kg bw/day.

Food Consumption: Food consumption was unaffected by treatment at 15, 50 or 150 mg/kg bw/day.

Water Consumption: Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Post Mortem Studies: No macroscopic abnormalities were detected for parental females at 15, 50 or 150 mg/kg bw/day.

Litter Responses

Litter Data and Litter Placental and Fetal Weights: The number of implantations, subsequent embryofetal survival and litter size, sex ratio and mean fetal, litter and placental weights on Day 20 of gestation were unaffected by maternal treatment at 15, 50 or 150 mg/kg bw/day.

Fetal Examination

There was no effect of maternal treatment on morphological development of the fetuses at 15, 50 or 150 mg/kg bw/day.

Conclusion

The No Observed Effect Level (NOEL) for the pregnant females and the survival, growth and embryofetal development of the offspring was considered to be 150 mg/kg bw/day.