[No public or meaningful name is available]

Toxicity to reproduction

Administrative data

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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

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.

Test material

Test material information

Constituent 1

Reference substance name:

Amides, C18, branched and linear

Molecular formula:

not applicable (UVCB substance)

IUPAC Name:

Amides, C18, branched and linear

Test material form:

solid: pellets

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.

Test animals

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Mid dose

Doses / concentrations 4

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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

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.