[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Other studies: new data available. Data produced to comply with EPA (USA data request)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 March 2017 to 21 July 2017

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The study was conducted in accordance with the applicable sections of the United Kingdom
Animals (Scientific Procedures) Act 1986, Amendment Regulations 2012 (the Act).

Organization for Economic Co-operation and Development: Testing of Chemicals
(Guideline 421; Reproduction/Developmental Toxicity Screening Test;
29 July 2016).
The study was conducted in accordance with the requirements of current, internationally
recognized Good Laboratory Practice Standards.

Specific details on test material used for the study:

The required amount of test item was ground in a mortar
using a pestle to a fine powder and mixed with a small
amount of the vehicle to form a paste. Any agglomerates
were broken down. Further amounts of vehicle were
gradually added and mixed to produce a smooth, pourable
suspension. The suspension was transferred to a
measuring cylinder which had been wetted with vehicle,
the mortar was rinsed with vehicle and this was added to
the measuring cylinder. Vehicle was added to achieve the
final volume and the suspension was transferred to a
beaker and mixed using a high shear homogenizer. The
suspension was transferred to the final containers, via
syringe whilst magnetically stirring.
A series of suspensions at the required concentrations were
prepared by dilution of individual weighings of the test
item.

Species:

rat

Strain:

Wistar

Remarks:

RccHan™;WIST rat.

Details on species / strain selection:

Strain/Species RccHan™;WIST rat.
Supplier Envigo RMS (UK) Ltd.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Number & Sex
Number of animals ordered 44 males and 48 females.
Spare animals were removed from the study room after
treatment commenced.
Acclimatization
Duration of acclimatization Males: Eight days before commencement of treatment.
Females: 22 days before commencement of treatment.
Age of the animals at the
start of the study
Males 86 to 92 days old.
Females 100 to 106 days old.
Weight range of the animals
at the start of the study
Males 298 to 361 g.
Females 199 to 238 g.
Allocation On arrival and non-selective allocation to cages.
Estrous cycles were evaluated prior to treatment. After
14 days evaluation, animals that failed to exhibit typical 4-5
day cycles were not allocated to the study.
On Day 1 of study all animals were weighed and body
weights were reviewed by Study Management before
treatment commenced to ensure variations in body weight of
animals did not exceed 20% of the mean for each sex.
Groups were adjusted to reduce inter-/intra-group variation.
Identification of animals Each adult animal was assigned a number and identified
uniquely within the study by a tail tattoo before Day 1 of
treatment. The offspring were numbered individually within
each litter on Day 1 of age, using a toe tattoo.
Identification of cages Each cage label was color-coded according to group and
was numbered uniquely with cage and study number, as
well as the identity of the occupant(s).
Replacement
Before the commencement of treatment, study allocation was revised to reduce inter/intra
group body weight variation by replacement of animals with spares and moving animals
within groups. Any individuals rejected during the acclimatization period were replaced
with spare animals of suitable weight from the same batch.
Replacement before
treatment
Irregular estrous cycle two females
Body weight range extremes one male and two females
Environmental
Rodent facility Limited access - to minimize entry of external biological
and chemical agents and to minimize the transference of
such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not
recirculated.
Temperature and relative
humidity
Monitored and maintained within the range of 20-24ºC and
40-70%.
There were no deviations from these ranges.
Lighting Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.
Accomodation
Cages Cages comprised of a polycarbonate body with a stainless
steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the
acclimatization, pre-pairing (treatment), gestation, littering
and lactation periods.
Grid bottomed cages were used during pairing. These were
suspended above absorbent paper which was changed daily
during pairing.
Cage distribution The cages were distributed on the racking to equalize, as far
as possible, environmental influences amongst the groups.
Bedding Solid bottom cages contained softwood based bark-free
fiber bedding, which was changed at appropriate intervals
each week.
Number of animals per cage
Pre-pairing up to five animals of one sex
Pairing one male and one female
Males after mating up to five animals
Gestation one female
Lactation one female + litter
Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage
throughout the study (except during pairing and lactation)
and replaced when necessary.
Polycarbonate shelter Provided to each cage throughout the study (except during
pairing and lactation) and replaced at the same time as the
cages.
Diet
Diet SDS VRF1 Certified pelleted diet.
A sample (100g) of each batch of diet used was retained
within Pharmacy (frozen -10 to -30ºC) until finalization of
the report. Samples were discarded after finalization of the
report.
The diet contained no added antibiotic or other
chemotherapeutic or prophylactic agent.
Availability Non-restricted.
Water
Supply Potable water from the public supply via polycarbonate
bottles with sipper tubes. Bottles were changed at
appropriate intervals.
Availability Non-restricted.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

BP Grade

Details on exposure:

Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed
directly.
Route Oral gavage using a suitably graduated syringe and a rubber
catheter inserted via the mouth.
Treated at Constant doses in mg/kg/day.
Volume dose 8 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body
weight.
Control (Group 1) Vehicle at the same volume dose as treated groups.
Frequency Once daily at approximately the same time each day.
Animals were not dosed if parturition was in progress at the
scheduled time of administration.
Formulation A daily record of the usage of formulation was maintained
based on weights. This balance was compared with the
expected usage as a check of correct administration. No
significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and
throughout the dosing procedure.

Details on mating procedure:

Pairing commenced After a minimum of two weeks of treatment.
Male/female ratio 1:1 from within the same treatment groups.
Duration of pairing Up to two weeks.
Daily checks for evidence
of mating
Ejected copulation plugs in cage tray and sperm in the vaginal
smear.
Day 0 of gestation When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and
evidence of mating.

Analytical verification of doses or concentrations:

yes

Remarks:

Validated method

Details on analytical verification of doses or concentrations:

The homogeneity and stability was confirmed for Amides, C18, branched and linear in
Arachis oil formulations at nominal concentrations of 1.25 mg/mL and 125 mg/mL during
distribution between the bottles, during magnetic stirring for 2 hours and at ambient
temperature storage for 1 day.
The mean concentrations of Amides, C18, branched and linear in test formulations analyzed
for the study were within ±16% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

Route Oral gavage using a suitably graduated syringe and a rubber
catheter inserted via the mouth.
Treated at Constant doses in mg/kg/day.
Volume dose 8 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body
weight.
Control (Group 1) Vehicle at the same volume dose as treated groups.
Frequency Once daily at approximately the same time each day.
Animals were not dosed if parturition was in progress at the
scheduled time of administration.
Formulation A daily record of the usage of formulation was maintained
based on weights. This balance was compared with the
expected usage as a check of correct administration. No
significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and
throughout the dosing procedure.

Frequency of treatment:

Route Oral gavage using a suitably graduated syringe and a rubber
catheter inserted via the mouth.
Treated at Constant doses in mg/kg/day.
Volume dose 8 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body
weight.
Control (Group 1) Vehicle at the same volume dose as treated groups.
Frequency Once daily at approximately the same time each day.
Animals were not dosed if parturition was in progress at the
scheduled time of administration.
Formulation A daily record of the usage of formulation was maintained
based on weights. This balance was compared with the
expected usage as a check of correct administration. No
significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and
throughout the dosing procedure.

Details on study schedule:

Study initiation (Study Plan
signed by Study Director)
17 March 2017
Experimental start date
(Pre-study chemistry)
28 February 2017
Animal arrival
Females
Males
5 April 2017
19 April 2017
Treatment commenced 27 April 2017
F0 pairing commenced 11 May 2017
F0 necropsy
Males
Females
30 May 2017
15 to 22 June 2017
Experimental completion
date
21 July 2017

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

10 males and 10 females per dose group

Control animals:

yes, concurrent no treatment

Details on study design:

Study Design and Identity of Treatment Groups

Males and females dose 15 days prior to mating. Males sacrificed during week 5 of treatment. Birth taken as day 1 of lactation. Females sacrificed at Day 13 of lactation

Positive control:

No, vertebrate study

Parental animals: Observations and examinations:

Signs associated with dosing

Clinical Observations
Clinical observations are presented for each animal that showed signs, providing detail of the
type of sign, day of occurrence and information on the duration of the sign applicable.

Body Weight
Group mean values and SD were calculated from individual body weight data on each
recorded occasion. For the offspring, litter mean body weight (+ SD) was calculated
separately for males and females and the group mean values derived from the individual litter
values.
Group mean weight changes were calculated from the weight changes of individual animals.
Offspring body weight change was calculated relative to Day 1 of age.
Body weights were plotted graphically with respect to the start of dosing or the start of the
relevant period.


Food Consumption
Group mean food consumptions and standard deviations were derived from unrounded cage
values.


gestational length
Parturition
Thyroid hormaone analysis

Oestrous cyclicity (parental animals):

Estrous Cycle
The percentage females showing the following classifications of estrous cycles before
treatment commenced and during treatment are presented:
Regular: All observed cycles of 4 or 5 days (divided into cycles of 4, 4 and
5 and 5 days)
Irregular: At least one cycle of 2, 3 or 6 to 10 days
Acyclic: At least 10 days without estrus
Vaginal smearing prior to termination is presented in terms of numbers of females that
showed estrus during this period and the cycle stage at termination.

Pre-Coital Interval
Individual intervals were tabulated for females only, for the time elapsing between initial
pairing and mating. Percentage of females with pre-coital intervals calculated for durations
of 1-4, 5-8, 9-12 and 13-14 days of pairing.

Sperm parameters (parental animals):

Ejected copulation plug and sperm in the vaginal smear

Litter observations:

Litter Size
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and
live at Days 1, 4, 7 and 13 of age. Group mean litter size and SD were calculated from the
individual litter values.

Sex Ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring
on Days 1, 4 (before and after blood sampling) and 13 of age.

Bodyweights
Nipple/areola count

Postmortem examinations (parental animals):

Abnormalities, Epididymides, Ovaries, Prostate, Seminal vesicles, Testes, Thyroids, Uterus with cervix and oviducts, Vagina
Female: implantation sites in each uterine horn, mammary tissue appearance

Postmortem examinations (offspring):

Macroscopic examination; in particular external genitalia

Statistics:

Statistical analyses were performed on the majority of data presented and results of these
tests, whether significant or non-significant, are presented on the relevant tables. For some
parameters, including estrous cycles before treatment, stage of estrous cycle at termination,
gestation index and viability index the similarity of the data was such that analyses were not
considered to be necessary.
All statistical analyses were carried out separately for males and females using the individual
animal as the basic experimental unit. For litter/fetal findings the litter was taken as the
treated unit and the basis for statistical analysis and biological significance was assessed with
relevance to the severity of the anomaly and the incidence of the finding within the
background control population.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs or signs observed following dose administration that were
considered to be related to treatment with Amides, C18, branched and linear.
Chin rubbing and salivation was observed in one female receiving 1000 mg/kg/day on one
occasion and salivation was observed on one occasion for one female receiving
100 mg/kg/day following dose administration.
There were no unscheduled deaths.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean bodyweight and bodyweight gain prior to pairing and during gestation for animals
receiving 100, 300 or 1000 mg/kg/day was similar to that of the Controls and were
considered to be unaffected by treatment with Amides, C18, branched and linear.
Mean bodyweight gain was slightly higher for treated females during late lactation and
overall when compared with Controls, but a dose trend was not apparent.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption prior to pairing and during gestation was similar to Controls and
considered unaffected by treatment with Amides, C18, branched and linear.
Group mean food intake was higher during lactation for females receiving 100 or
1000 mg/kg/day, when compared with Controls. Group mean food intake for females
receiving 300 mg/kg/day was similar to that of the Controls during the lactation period.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis
All samples taken from adult males at termination and day 13 of age male and female
offspring from animals in Groups 1 to 4 had concentrations that were in the region of the
endogenous levels observed in the control matrix used to prepare the QC samples.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The microscopic examination performed in Week 5 of treatment for males or on Day 13 of
lactation for females revealed no test item related lesions.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle
and the integrity of the various cell types present within the different stages. No cell or stage
specific abnormalities were noted. There were no test item related findings seen in the
ovaries following the qualitative examination.
The incidence and distribution of all findings were considered to be unrelated to treatment.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycles, pre-coital interval and mating performance and fertility was considered
unaffected by treatment with Amides, C18, branched and linear, when compared with
Controls.
One female receiving 1000 mg/kg/day was acyclic during treatment, however once in pairing,
she mated at the first possible opportunity (first day of pairing). At termination, all females
were in diestrus.
Gestation length was within the expected time frame 22-23 days for all animals, with the
exception of one Control female who had a gestation length of 24 days; gestation length and
gestation index were considered unaffected by treatment at any dose level.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Estrous cycles, pre-coital interval and mating performance and fertility was considered
unaffected by treatment with Amides, C18, branched and linear, when compared with
Controls.
One female receiving 1000 mg/kg/day was acyclic during treatment, however once in pairing,
she mated at the first possible opportunity (first day of pairing). At termination, all females
were in diestrus.
Gestation length was within the expected time frame 22-23 days for all animals, with the
exception of one Control female who had a gestation length of 24 days; gestation length and
gestation index were considered unaffected by treatment at any dose level.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Clinical signs:

no effects observed

Description (incidence and severity):

The distribution of signs at physical examination showed no relationship to parental
treatment.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Litter size up to Day 13 of age was unaffected by treatment with Amides, C18, branched and
linear. The number of females raising their litters to Day 13 of age was 9, 10, 8 and 10 for
Control, 100, 300 and 1000 mg/kg/day, respectively. One Control female and one female
receiving 300 mg/kg/day were not pregnant (1F 45 and 3F 68), and total litter loss was
recorded in one female treated with 300 mg/kg/day (3F 66) which was considered unrelated
to treatment.
Group mean percentage of post implantation survival index, live birth index, viability index
and lactation index were not affected by treatment with Amides, C18, branched and linear.
The Control group mean litter size, percentage for post implantation survival index and live
birth index was slightly lower than that of the Historical Control Data range (See Annex 5).
There were no effects on sex ratio that were associated to treatment with Amides, C18,
branched and linear.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean offspring bodyweight and mean bodyweight gain for males and females offspring of
parents treated with Amides, C18, branched and linear, at 100, 300 or 1000 mg/kg/day were
not affected by treatment when compared with Controls.
The slightly lower growth of male offspring in all treated groups and female offspring in the
300 or 1000 mg/kg/day groups, when compared with Controls is considered to reflect the
slightly higher litter size in these groups which was unrelated to treatment.

Other effects:

no effects observed

Description (incidence and severity):

There was no conclusive effect on ano-genital distance in the offspring of parents treated with
Amides, C18, branched and linear.
No nipples were observed in male offspring of parents treated with Amides, C18, branched
and linear.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Key result

Reproductive effects observed:

no

Conclusions:

The purpose of this study was a screening test for reproductive/development effects, with
administration of the test item Amides, C18, branched and linear, an additive, by oral gavage
administration for at least four weeks.
Three groups of ten male and ten female rats received Amides, C18, branched and linear at
doses of 100, 300 or 1000 mg/kg/day by oral gavage administration. Males were treated
daily for 15 days before pairing, up to necropsy after a minimum of four consecutive weeks.
Females were treated daily for 15 days before pairing, throughout pairing, gestation and until
Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on
Day 13 of lactation. The F1 generation received no direct administration of the test item; any
exposure was in utero or via the milk. A similarly constituted Control group received the
vehicle; Arachis oil BP, at the same volume dose as treated groups.
During the study, clinical condition, body weight, food consumption, estrous cycles,
pre-coital interval, mating performance, fertility, gestation length, organ weight and
macroscopic pathology and histopathology investigations were undertaken.
The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance
and nipple counts (males only), and macropathology for all offspring were also recorded.
Results
The mean concentrations of Amides, C18, branched and linear in test formulations analyzed
for the study were within ±16% of nominal concentrations, confirming accurate formulation.
Administration of Amides, C18, branched and linear at dose levels of 100, 300 and
1000 mg/kg/day was well tolerated with no effects that could be attributed to treatment on
clinical condition, oestrous cycles, organ weights, macroscopic examination or
histopathology. In addition there were no effects on pre-coital interval, mating performance,
fertility, gestation length, offspring survival or development. Bodyweight performance and
food consumption was unaffected by treatment before pairing and during gestation, but
bodyweight gain was slightly higher during lactation for treated females, and food intake was
higher for females receiving 100 or 1000 mg/kg/day, when compared with Controls.
There was no test item related finding seen in this study, following the histopathological
examination of testes and epididymides in males, ovaries of females or abnormalities in both
sexes given Amides, C18, branched and linear.
Conclusion
It was therefore concluded that in the absence of any evidence for general systemic toxicity
or effects on reproductive performance/offspring development that the
no-observed-adverse-effect-level was 1000 mg/kg/day.

Executive summary:

The no-observed-adverse-effect-level was 1000 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Key Study

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, 8.7.1., column 2, a screening test for toxicity to reproduction is not required as a test for prenatal developmental toxicity (Annex IX, 8.7.2) is available for the structurally-related substance (Z)-docos-13-enamide (CAS 112-84-5, erucamide), which is used as basis for read-across. On the basis of this prenatal developmental toxicity study no test item-related toxicological findings in dams or fetuses were revealed. Therefore, no adverse effects are to be expected for Amides, C18, branched and linear, either.

 

According to Regulation (EC) No 1907/2006, Annex IX, 8.7.3, column 1, a 2-generation study for reproductive toxicity is not indicated unless repeated dose toxicity studies demonstrate adverse effects on reproductive organs or tissues. A subchronic repeated dose toxicity study (90-days) is available for the structurally-related substance (Z)-docos-13-enamide (CAS 112-84-5, erucamide) with additional focus on assessment of fertility parameters and effects on reproductive organs. No test item-related effect on epididymal sperm motility or testicular sperm count was noted at the end of the treatment of this study. The test substance had no biologically significant effect on the estrous cycle analyzed 4, 8 and 12 weeks after the first administration. In addition, histopathology demonstrated no effects on reproductive organs. Thus, a 2-generation study for reproductive toxicity with Amides, C18, branched and linear is not indicated on the basis of no observed adverse effects of the structurally-related substance.

 

A sub chronic developmental screening study is now available and was produced in response to a data requirement for the US EPA. The NOAEL was 1000 mg/kg bodyweight and supports the current read-across data well.

Short description of key information:
Waiving - Toxicity to reproduction screening
Waiving - Toxicity to reproduction 2-generation study

Key Study - sub chronic developmental screening study

Effects on developmental toxicity

Description of key information

Oral (OECD 414): NOAEL (developmental) >= 1000 mg/kg bw/day

Oral (OECD 421): NOAEL (reproduction & developmental) 1000 mg/kg bodyweight
Inhalation: no data available
Dermal: no data available

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:

25 Sep 2014 - 11 May 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted according to the appropriate OECD test guideline and in compliance with GLP. 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)

Version / remarks:

(2001)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit

Limit test:

no

Species:

rat

Strain:

other: Wistar rats, Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 11 - 12 weeks (females), 19 – 20 weeks (males)
- Weight at study initiation: 200 – 243 g (females), 390 – 522 g (males)
- Housing: The animals were kept individually in IVC cages (except during the pre-mating period when females were kept in groups of two animals and mating period when two females were paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 02102140509)
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 1526), ad libitum
- Water: Sulphur acidified tap water, ad libitum
- Acclimation period: at least five days

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

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was ground to a fine powder with the help of a mortar and pestle. The powdered test item was weighed into a tarred plastic vial on a precision balance and the vehicle corn oil was added to give the appropriate final concentration of the test item. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration. The test item formulation was prepared freshly on each administration day before the administration procedure.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline.
- Concentration in vehicle: 14.29 mg/mL (low dose, LD), 42.86 mg/mL (medium dose, MD), 142.86 mg/mL (high dose, HD)
- Amount of vehicle (if gavage): 7 mL/kg bw/day
- Lot/batch no. (if required): MHBP7039V

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of 13-Docosenamide, (Z)- was performed using GC with FID detection. Formulation analysis was performed on samples of all dose groups collected at various intervals during the study. Concentration analysis of formulation samples was performed in the first and the last study week for all dose groups. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 98.2%, 96.3% and 110.8% of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations did not differ from nominal concentrations by more than 20%. Stability of formulation samples was investigated in study week 1 based on concentration in the samples taken from the LD, MD and HD dose groups. After 3 hours storage at room temperature the recovery compared to the starting value was between 90.1% and 96.3%. All samples were stable, as concentration after storage did not differ from the start value by more than 20%. Homogeneity of formulation samples was determined in the first and the last study week for the LD, MD and HD dose groups. The mean recovery observed for the LD dose group was 113.5% and 109.2% of the nominal value, for the MD dose group 99.8% and 113.8% of the nominal value and for the HD dose group 107.0% and 110.1% of the nominal value. The coefficients of variation (COV) of the different sampling locations (top, middle, bottom) were 13.6% and 10.8% in the LD dose group, 8.9% and 13.0% in the MD dose group and 4.7% and 4.5% in the HD dose group. All samples were homogenous, as COV was below or equal to the acceptance criterion of 20%.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: Females were paired for cohabitation in batches in order to regularise the number of animals for terminal sacrifice on a particular day. The subsequent morning and each morning thereafter, the vaginal smear of each female was checked until positive evidence of mating was confirmed.
- Proof of pregnancy: The day on which sperms were observed in the vaginal smear was considered as gestation day ‘0’. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that the mean body weights were comparable to each other.
- Any other deviations from standard protocol: The mating resulted in 23 sperm-positive females in the control and 24 each in the LD, MD and HD group. Non sperm-positive females were excluded from the study without any further observations.

Duration of treatment / exposure:

Day 5 - 19 of gestation

Frequency of treatment:

daily, 7 days/week

Duration of test:

Until Day 20 of gestation / post mating

No. of animals per sex per dose:

22 pregnant females (control)
21 pregnant females (LD)
23 pregnant females (MD)
20 pregnant females (HD)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: According to the results of the dose range finding study (BSL study No. 143124) and in consultation with the sponsor doses of 100, 300 and 1000 mg/kg bw/day were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily, preferably at the same time each day, animals were observed for spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Sperm-positive females on Gestation Days 0, 5, 8, 11, 14, 17 and 20. Males were only weighed once prior to mating.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption of pregnant females was measured on Gestation Day 5, 8, 11, 14, 17 and 20.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: At the time of termination or death during the study, the dam (presumably pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Uteri that appeared non-gravid were further examined by staining with 10% ammonium sulphide solution to confirm the non-pregnant status.

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
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

A statistical assessment of the body weight and food consumption results, prenatal parameters and litter weight data was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Fetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using a Fisher’s exact test. Litter incidence was the primary unit for the statistical analysis and interpretation. The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Historical control data:

Historical control data from the Test Facility were not provided.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
All animals survived to the scheduled necropsy and no mortality was observed in this study. No test item-related clinical signs were observed in any of the treated groups. A few spontaneous clinical signs were observed in few animals, but no effect on overall health, body weight and food consumption was observed in these animals. The mean body weight and body weight gain were unaffected by test item administration during the gestation period when compared with the controls. Throughout the treatment period, body weights and body weight gain increased as the study progressed and were within the normal range of variation for this strain. No treatment-related effect on food consumption was observed during the treatment period. No treatment-related effects were observed in terminal body weight, gravid uterine weight, adjusted maternal weight, number of corpora lutea, implantations, resorptions, percent preimplantation loss and percent postimplantation loss. There were no statistically significant differences. Successful mating resulted in 21/24 pregnancies in the LD group, 23/24 in the MD group and 20/24 in the HD group, compared to 22/23 pregnancies in the control group. Low pregnancy rate in the LD and HD group was considered to be a biological variation and of no toxicological relevance. No gross pathological changes were observed during the macroscopic examination of females of the control, the LD and the MD group. However, in the HD group fluid distension of the uterus was observed in one female, and in another female white discoloured areas on the liver, white to yellow discolouration of the left side of the oesophagus/trachea, yellow fluid content in the thoracic cavity and enlarged adrenal glands were observed at necropsy. Due to the isolated incidence in only two animals, these gross pathological findings were considered to be spontaneous in nature and as such not considered to be a systemic effect due to test item administration.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Statistical analysis of litter data revealed no treatment-related effects on group mean number of live fetuses and dead fetuses, number of male and female fetuses, total number of fetuses or sex ratio when compared with the controls. No treatment-related effect on per litter data parameters including group litter mean weight, total litter weight and male litter weight was observed. However, statistically significantly lower female litter weight was observed in the LD group when compared with the controls. Due to lack of dose dependency and consistency, this effect on female litter weight was considered to be of no toxicological relevance. There were no external abnormalities considered to be of toxicological relevance in any of the dose groups. The statistical analysis showed no significant differences to the control group. A range of visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower compared to the controls, and litter incidences were statistically insignificant except significantly lower discolouration of the dexter lobe of the liver in the MD group when compared to the controls. As the observed finding was minor and lacked dose dependency and consistency, no toxicological significance can be attributed to it; this finding was considered to be spontaneous in nature. All remaining fetal visceral findings revealed no significant alterations compared to the control group. Craniofacial examination by a razor blade serial sectioning technique revealed few findings (slightly dilated right or both lateral ventricles, right or left retinal fold and slightly dilated 3rd lateral ventricle) at frequencies generally comparable to or in some cases slightly higher or lower compared to the controls. Statistical analysis of the data revealed no significant effect in any of these findings as compared to the control group. Therefore, these findings are considered to be not treatment related and spontaneous in nature. Skeletal examination of Alizarin red stained fetuses revealed a range of abnormalities in the treated groups which were of a type comparable to the control group or which occurred at an incidence either comparable to or lower or sometimes higher than in the control group. A statistically significant increase in the litter incidence of incomplete ossification of the interparietal bone in the LD group was observed when compared to the control group. Due to lack of dose dependency and consistency this findings was not assumed to be test item related. All remaining skeletal examinations revealed no significant alterations compared to the control group.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed

Abnormalities:

not specified

Key result

Abnormalities:

no effects observed

Localisation:

other: No effects observed

Developmental effects observed:

not specified

Table 1: Litter data – summary

Group		Mean litter weight (g)	Number of males	Number of females	Live foetuses	Dead foetuses	Sex ratio	Total litter weight (g)	Male litter weight (g)	Female litter weight (g)
C	Mean	3.86	5.18	5.68	10.86	0.00	0.98	42.26	20.46	21.80
	SD	0.58	1.97	1.55	2.38	0.00	0.50	12.79	8.71	7.87
	N	22	22	22	22	22	22	22	22	22
LD	Mean	3.67	5.33	4.62	9.95	0.00	1.40	35.84	20.00	15.84*
	SD	0.44	2.24	2.16	3.23	0.00	1.12	10.99	9.07	6.63
	N	21	21	21	21	21	20 a)	21	21	21
MD	Mean	3.83	5.26	5.17	10.43	0.00	1.18	39.67	20.27	19.40
	SD	0.59	1.66	1.40	1.44	0.00	0.77	6.37	5.86	6.17
	N	23	23	23	23	23	23	23	23	23
HD	Mean	3.83	4.95	5.70	10.60	0.05	1.01	40.34	19.44	20.90
	SD	0.51	1.64	2.15	2.68	0.22	0.55	10.25	6.86	7.75
	N	20	20	20	20	20	20	20	20	20

Asterisks indicate significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

C: Control

LD: Low dose

MD: Medium dose

HD: High dose

N: Number of pregnant females (dams)

a): One dam in the LD group with no female pup, the animal was therefore excluded from sex ratio calculation

Table 2: Fetal skeletal findings – summary

Skeletal findings		Group
		1	2	3	4
		C	LD	MD	HD
Skull interparietal incomplete ossification	No of Incidences	21	30	30	19
	Total No of Observed Foetuses	125	109	123	104
	% Foetus Incidence	16.80	27.52	24.39	18.27
	No of Litters with at least 1 incidence	12	15*	14	8
	Total No of Observed Litters	22	21	23	20
	% Litter Incidence	54.55	71.43	60.87	40.00

Asterisks indicate significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

C: Control

LD: Low dose

MD: Medium dose

HD: High dose

Table 3: Fetal visceral findings – summary

Visceral findings		Group
		1	2	3	4
		C	LD	MD	HD
Liver dexter lobe discolored (dark)	No of Incidences	0	0	6	1
	Total No of Observed Foetuses	114	100	117	102
	% Incidence of Abnormality	0.00	0.00	5.13	0.98
	No of Litters with at least 1 incidence	0	0	5*	1
	Total No of Observed Litters	22	21	23	20
	% Litter Incidence	0.00	0.00	21.74	5.00

Asterisks indicate significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

C: Control

LD: Low dose

MD: Medium dose

HD: High dose

Conclusions:

On the basis of this prenatal developmental toxicity study in pregnant female Wistar rats with 13-Docosenamide, (Z)- at dose levels of 100, 300, and 1000 mg/kg bw/day administered from gestation days 5 to 19 no test item related toxicological findings in dams or fetuses were revealed. Under the conditions of the study, 1000 mg/kg bw/day was considered as no observed adverse effect level (NOAEL) for both maternal and embryo-fetal toxicity of 13-Docosenamide, (Z)-.

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

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

Effect on developmental toxicity: via oral route

No data on developmental toxicity is available for Amides, C18, branched and linear. Therefore, read across from the structurally related substance (Z)-docos-13-enamide (erucamide, CAS 112-84-5) was applied. In the available key study (BSL, 2015) the test substance (Z)-docos-13-enamide (CAS 112-84-5) was investigated for prenatal developmental toxicity after repeated oral administration conducted according to OECD TG 414, and in compliance with GLP. Groups of 21 (low dose), 23 (medium dose) and 20 (high dose) pregnant female Crl:WI(Han) rats per dose were administered doses of 100, 300 and 1000 mg/kg bw/day via oral gavage. Animals treated with the vehicle (corn oil) served as controls. Treatment was carried out once daily during the gestation period between Day 5 to Day 19. Animals were observed for mortalities and clinical signs, and detailed clinical observations were performed twice daily (except weekends). Body weight was recorded on gestation days 0, 5, 8, 11, 14, 17 and 20. Upon sacrifice on gestation day 20 the dam (presumably pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. The ovaries and uterine content were examined after termination. Fetuses were subjected to external and either soft tissue and head or skeletal examination. All animals survived to the scheduled necropsy, and no mortality was observed in this study. No test item-related clinical signs were observed in any of the treated groups. A few spontaneous clinical signs were observed in few animals, but no effect on overall health, body weight and food consumption was observed in these animals. The mean body weight and body weight gain were unaffected by test item administration during the gestation period when compared with the controls. No treatment-related effects were observed in terminal body weight, gravid uterine weight, adjusted maternal weight, number of corpora lutea, implantations, resorptions, percent preimplantation loss and percent postimplantation loss. There were no statistically significant differences. No gross pathological changes were observed during the macroscopic examination of females of the control, the LD and the MD group. However, in the HD group fluid distension of the uterus was observed in one female, and in another female white discoloured areas on the liver, white to yellow discolouration of the left side of the oesophagus/trachea, yellow fluid content in the thoracic cavity and enlarged adrenal glands were observed at necropsy. Due to the isolated incidence in only two animals, these gross pathological findings were considered to be spontaneous in nature and as such not considered to be a systemic effect due to test item administration. In conclusion, no maternal toxic effects were observed after administration of the test substance. Statistical analysis of litter data revealed no treatment-related effects on group mean number of live fetuses and dead fetuses, number of male and female fetuses, total number of fetuses or sex ratio when compared with the controls. No treatment-related effect on per litter data parameters including group litter mean weight, total litter weight and male litter weight was observed. However, statistically significantly lower female litter weight was observed in the LD group when compared with the controls. Due to lack of dose dependency and consistency, this effect on female litter weight was considered to be of no toxicological relevance. There were no external abnormalities considered to be of toxicological relevance in any of the dose groups. A range of visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower compared to the controls, and litter incidences were statistically insignificant except significantly lower discolouration of the dexter lobe of the liver in the MD group when compared to the controls. As the observed finding was minor and lacked dose dependency and consistency, no toxicological significance can be attributed to it; this finding was considered to be spontaneous in nature. All remaining fetal visceral parameters revealed no significant alterations compared to the control group. Craniofacial examination by a razor blade serial sectioning technique revealed few findings (slightly dilated right or both lateral ventricles, right or left retinal fold and slightly dilated 3rd lateral ventricle) at frequencies generally comparable to or in some cases slightly higher or lower compared to the controls. Statistical analysis of the data revealed no significant effect in any of these findings as compared to the control group. Therefore, these findings are considered to be not treatment- related and spontaneous in nature. Skeletal examination of Alizarin red stained fetuses revealed a range of abnormalities in the treated groups which were of a type comparable to the control group or which occurred at an incidence either comparable to or lower or sometimes higher than in the control group. A statistically significant increase in the litter incidence of incomplete ossification of the interparietal bone in the LD group was observed when compared to the control group. Due to lack of dose dependency and consistency this findings was not assumed to be test item-related. All remaining skeletal examinations revealed no significant alterations compared to the control group. In summary, administration of (Z)-docos-13-enamide (CAS 112-84-5) at doses of 100, 300 or 1000 mg/kg bw/day by oral gavage to pregnant female Wistar rats from gestation days 5 to 19 resulted in significantly lower female litter weight as well as lower discolouration of the dexter lobe of the liver and significant increase in the litter incidence of incomplete ossification of the interparietal bone. These findings were considered to be not test item-related and of no toxicological relevance due to lack of dose dependency and consistency. Therefore, both the maternal and embryo-fetal No-Observed-Adverse-Effect-Levels (NOAEL) were considered to be 1000 mg/kg bw/day.

Justification for selection of Effect on developmental toxicity: via oral route:
Hazard assessment is conducted by means of read-across from a structural analogue/surrogate. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose.

Justification for classification or non-classification

The available data on the developmental toxicity of a structurally related substance according to the criteria of 'ECHA Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals' do not meet the criteria for classification according to Regulation (EC) No 1272/2008 or Directive 67/548/EEC; therefore, Amides, C18, branched and linear is not expected to exert adverse effects, either, and the data are thus conclusive but not sufficient for classification.