Mono-, and di-(sec-hexadecyl)naphthalene

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No data available.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jun 2012 - May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted with no exceptions

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain/Species Crl:CD(SD) rat.
Supplier Charles River (UK) Ltd
Number of animals 128 males and 128 females.
Animals were identified littermates of four of one sex per
litter. 32 litters of 4 males and 32 litters of 4 females were
received and males were not related to females.
Duration of acclimatisation
for the F0 animals
12 days before commencement of treatment.
Age of the F0 animals at start
of treatment
34 to 40 days old.
Weight range of the F0
animals at the start of
treatment
Males: 131 g to 199 g
Females: 116 g to 168 g

Environmental control
Rodent facility Full barrier to minimise entry of external biological and
chemical agents and to minimise 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 19-23ºC and
40-70%.
Although conditions were occasionally outside the indicated
ranges, these deviations were minor and of short duration and
were not considered to have influenced the health of the
animals or the outcome of the study (see Deviations from
protocol section).
Lighting Artificial lighting, 12 hours light : 12 hours dark.

Animal accommodation and bedding
Cages Cages comprised of a polycarbonate body with a stainless
steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used throughout the
study except during pairing.
Grid bottomed cages (polypropylene) were used during
pairing. These were suspended above absorbent paper which
was changed daily during pairing.
Cage distribution The cages constituting each group were dispersed in batteries
so that possible environmental influences arising from their
spatial distribution were equilibrated, as far as was
practicable.
Bedding Solid bottom cages contained wood based bedding, which was
changed at appropriate intervals each week.

Number of animals per cage Pre-pairing (acclimatisation
and after selection)
up to 4 animals
Pairing one male and one female animal
Males to termination up to 4 animals
Females after mating (from
Day 0 after mating)
one animal
Females during littering
(from Day 20 after mating)
one animal + litter
Females to termination
(after weaning)
up to 4 animals
Offspring maturation (from
weaning until selection)
litter
Environmental enrichment
Aspen chew 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 supply
Diet SDS VRF1 Certified powdered diet.
The diet contained no added antibiotic or other
chemotherapeutic or prophylactic agent.
Availability Non-restricted.
Water supply
Supply Potable water from the public supply via polycarbonate
bottles with sipper tubes. Bottles were changed at
appropriate intervals.

Route of administration:

oral: feed

Vehicle:

other: Powdered laboratory animal diet (SDS VFR1 Certified).

Details on exposure:

2.2.3 Formulation
The study substance was incorporated into the basal diet to provide the required concentrations
by dilution of an appropriate premix.
The required amount of [study substance] was added to an approximately equal amount of fine
sieved diet (355μm) by gentle stirring, using a spoon. An amount of sieved diet (355μm)
approximately equal to the weight of the mixture was then added and the mixture stirred until
it appeared visibly homogeneous. The doubling-up process was repeated until approximately
half the pre-mix weight was achieved. This mix was then ground using a mechanical grinder
and made up to the required weight using the remaining coarse diet from the sieving process.
At this stage, the mixture was stirred then mixed in a Turbula mixer for 100 cycles at 16 rpm
to ensure all the study substance was dispersed in the diet.
Aliquots of the appropriate premix were then diluted with further diet to produce the required
dietary concentrations for feeding to the animals; each batch of treated diet was mixed for a
further 100 revolutions at 16 rpm in the Turbula mixer.
The homogeneity and stability of formulations during storage were confirmed as part of
another study (Huntingdon Life Sciences Study Number: HJL0157). On that study,
homogeneity and stability were confirmed following storage at ambient temperature
(nominally 21 °C) for 22 days.
All diets were prepared weekly at the Huntingdon Research Centre and transported to the Eye
Research Centre for feeding to the animals.
Detailed records of compound usage were maintained. The amount of study substance
necessary to prepare the formulated diets and the amount actually used were determined on
each occasion of diet preparation. The difference between these amounts was checked before
the diets were dispensed.

Details on mating procedure:

F0 pairing commenced After 10 weeks of treatment.
F1 pairing commenced 10 weeks after selection (formal commencement of F1).
Male/female ratio 1:1 from within the same treatment groups (sibling pairing
was not permitted).
Duration of pairing Up to two weeks.
mating
Presence of ejected copulation plugs.
Vaginal smear - examined for the presence of spermatozoa
and the stage of the oestrous cycle.
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

Details on analytical verification of doses or concentrations:

2.2.4 Formulation analysis
The stability of a homogenous preparation of the study substance in the diet, at nominal
concentrations of 500 ppm and 15000 ppm, was demonstrated over a period of up to 22 days
at ambient temperature in Study number HJL0157. Stability was also confirmed following
storage at nominally -20°C for 22 days.
In this study, at specified intervals during treatment, the test formulations were analysed for
achieved concentration of the study substance (see Deviations from protocol section). On five
occasions, approximately 10 weeks apart, four samples (nominally 200 g) were taken from all
groups; two assays from each sample. Stored samples were retained as contingency for
analysis should any result require confirmation.
The method of analysis was an adaptation of a method supplied by the Sponsor; further
details on test procedures and results are presented in the Formulation Analysis Report
(Annex 2).

Duration of treatment / exposure:

F0: 10 weeks of treatment before pairing commenced. Males: 19 weeks. Females until d28 post-partum
F1: lifetime (10 weeks beginning after selection on PND 28, 10 weeks of treatment before pairing commenced. Males: 19 weeks. Females until d28 post-partum)
F2: until d28 post-partum

Frequency of treatment:

daily ad libitum

Remarks:

Doses / Concentrations:
1200 ppm
Basis:
nominal in diet
Refer to results section for achieved dose

Remarks:

Doses / Concentrations:
3500 ppm
Basis:
nominal in diet
Refer to results section for achieved dose

Remarks:

Doses / Concentrations:
12000 ppm
Basis:
nominal in diet
Refer to results section for achieved dose

No. of animals per sex per dose:

28 m/f in F0, 24 m/f in F1

Control animals:

yes, plain diet

Positive control:

none

Parental animals: Observations and examinations:

2.5.1 Clinical observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to
treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the
occupants. Any deviation from normal was recorded at the time in respect of nature and
severity, date and time of onset, duration and progress of the observed condition, as
appropriate.
During the acclimatisation period, observations of the animals and their cages were recorded
at least once per day.
Clinical signs
A detailed weekly physical examination was performed once each week for F0 animals and
for F1 animals selected for the formal F1 generation to monitor general health. Females also
had a detailed examination on Days 0, 5, 12, 18 and 20 after mating and on days 1, 7, 14, 21
and 25 of lactation.
Mortality
A viability check was performed near the start and end of each working day. Animals were
killed for reasons of animal welfare where necessary.
A complete necropsy was performed in all cases as described in section 2.6.
2.5.2 Bodyweight
Animals were weighed as follows:
F0 Males Day that treatment commenced (Week 0).
Each week.
At necropsy.
F0 Females Day that treatment commenced (Week 0).
Each week until mating was detected.
Days 0, 7, 14 and 20 after mating.
Days 1, 4, 7, 14, 21, 25 and 28 of lactation.

2.5.3 Food consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was
recorded was recorded as follows:
F0 males and females: Weekly until paired for mating. From these records the
mean weekly consumption per animal (g/rat/week) was
calculated for each cage.
F0 females: Days 0-6, 7-13 and 14-19 after mating and Days 1-3, 4-6,
7-13 and 14-20 of lactation. From these records the mean
daily consumption (g/rat/day) was calculated for each
animal.

2.5.6 Parturition observations and gestation length
Duration of gestation Time that elapsed between mating and commencement of
parturition.
Parturition observations From Day 20 after mating animals were checked three times
daily for evidence of parturition. The progress and
completion of parturition was monitored; numbers of live
and dead offspring were recorded and any difficulties
observed were noted.

Oestrous cyclicity (parental animals):

2.5.4 Oestrous cycles
Dry smears Smears were taken daily for 22 days before pairing, using
cotton swabs moistened with saline. The smears were
subsequently examined to establish the duration and
regularity of the oestrous cycle.
Wet smears After pairing with the male, daily smearing was continued
using pipette lavage, until evidence of mating was observed.
For four days before scheduled termination (nominally
Days 25 to 28 post partum) daily vaginal smears were taken
and used to determine the stage of the oestrous cycle at
termination.
For females whose litters had previously died, smears were taken on a theoretical
Days 25-28. Females that failed to litter or mate were retained and smeared for four days
starting on the day on which the first batch of ‘true’ Day 25 females started smearing, then
killed with that first batch of females.

Sperm parameters (parental animals):

2.6.1 Sperm analysis
Immediately after scheduled sacrifice of each F0 and selected F1 male, the left vas deferens,
epididymis and testis was removed and the epididymis and testis were weighed. Samples
were obtained from the right side for male 49.
The following tests were performed:
Sperm motility - all groups A sample of sperm was expressed from the vas deferens into
pre-warmed (37C) medium M199, which contained 0.5%
w/v bovine serum albumin (BSA Fraction V). A sample for
assessment was taken into a 100μm depth cannula by
capillary action and at least 200 sperm per animal analysed
using the Hamilton Thorne IVOS Computer Assisted Sperm
Analyser (CASA) version 12.3d. The percentages of motile
and progressively motile sperm were reported.
Sperm morphology - Groups 1
and 4
A 200L aliquot of the sperm/medium mixture (described
above) was diluted with 800L of 10% neutral buffered
formalin. After staining with nigrosine and eosin an
air-dried smear was prepared and examined by light
microscopy for the assessment of sperm morphology. At
least 200 sperm were assessed for each male of groups 1 and
4, where possible. The percentages of normal sperm and
abnormal sperm were reported
Sperm count - Groups 1 and 4 The left cauda epididymis of each male was weighed (right
cauda epididymis for male 49). The left cauda epididymis
for groups 2 and 3 were then frozen (except for group 2
male No. 328; see Deviations from protocol section). For
groups 1 and 4 and male 328, 10 mL of a mixture of 0.9%
saline, 0.01% merthiolate and 0.05% Triton X-100 (SMT)
was added and the cauda epididymis was homogenised for
at least one minute. An aliquot of this mixture was added to
a pre-prepared IDENT stain tube before being assessed for
sperm count using CASA. The concentration (Million/g)
and total number of sperm were reported. The data for male
328 was retained but has not been reported.
Homogenisation-resistant
spermatids count - Groups 1
and 4
The left testis of each male of groups 1 and 4 and male
2M 328 was homogenised for at least two minutes in 25 mL
of SMT. An aliquot of this mixture was added to a preprepared
IDENT stain tube before being assessed for
homogenisation-resistant spermatid count using CASA.
The concentration (Million/g) and total number of
spermatids were reported. Testes for groups 2 and 3 (except
male 328) were frozen. The data for male 328 was retained
but has not been reported.

Litter observations:

2.5.7 Records made during littering phase (F0 and F1 generation)
The records maintained were as follows:
Clinical signs All litters were examined at approximately 24 hours after
birth (Day 1 of age) and then daily thereafter.
Offspring identification On Day 1 of age each offspring was numbered individually
within each litter using a toe tattoo.
Litter size Daily records were maintained of mortality and consequent
changes in litter size from Days 1-21 of age.
On Day 4 of age, litters containing more than ten offspring
were reduced to ten by random culling, leaving, whenever
possible, five male and five female offspring in each litter.
Sex ratio of each litter Recorded on Days 1, 4 (before and after culling) and on
Day 21 of age.
Individual offspring
bodyweights
Recorded on Days 1, 4 (before culling), 7, 14 and 21 of age.
Weaning of offspring The dam was removed from the litter cage and offspring
were weaned on Day 21 of age.
Selection of offspring (F1
generation)
The selection of offspring to form the F1 generation was
made on Day 18 of age.

Postmortem examinations (parental animals):

The organs weighed, tissue samples fixed and sections examined microscopically are detailed
as follows:
Necropsy Histology Pathology
Tissue and regions to be examined Weigh Fix Light
microscopy
ADULT ANIMALS
Abnormalities * * *
Adrenals * * * *
Brain (cerebellum, cerebrum, midbrain) * * # #
Epididymides (caput, corpus and cauda) L+R † † †
Kidneys * * # #
Liver (section from all main lobes) * * # #
Mammary area – caudal b) # #
Ovaries d) L+R * * *
Pituitary * * * *
Prostate * * * *
Seminal vesicles (with coagulation gland) * * * *
Spleen * * # #
Testes L+R † † †
Thyroid with parathyroids a) * # #
Uterus with cervix and oviducts (separate section of each) * * * *
Vagina e) * * *
Target organs, if possible  * * * *
OFFSPRING c)
Abnormalities * $ $
Brain * * # #
Epididymides * # #
Ovaries * # #
Prostate * # #
Seminal vesicles * # #
Spleen * * # #
Testes * # #
Thymus * * # #
Uterus with cervix and oviducts * # #
Vagina * # #
L+R Bilateral organs weighed individually.
* Organs weighed, samples fixed or sections examined microscopically.
# Examined if effects suspected during the study.
† Only one examined - left testis and epididymis reserved for seminology.
$ Consideration was given to the offspring abnormalities recorded; only those deemed appropriate will be examined.
a) Weighed after partial fixation.
b) Females with total litter loss only
c) Procedures for only one male and one female offspring per litter (but abnormalities fixed from all offspring in
litter).
d) 5 sections cut at ca 100 micron intervals from the inner third of each ovary.
e) Section ca 5 mm from vulva.
The retained tissues were checked before disposal of the carcass.

2.6.2 Organ weights
Requisite organs were weighed for all F0 and F1 adult animals killed at scheduled intervals.
For F1 unselected and F2 offspring, one male and one female were selected at random from
each litter for organ weights.
Bilateral organs were weighed individually.

Statistics:

For bodyweight, food consumption, litter
size and survival indices, sexual maturation, organ weight and primordial ovarian follicle
counts data:
F1 approximate test used to determine if data is normally distributed (Bartlett's test for variance homogeneity was not significant at 1%).
If the F1 approximate test for monotonicity of dose-response was not
significant at the 1% level, Williams' test for a monotonic trend was applied.
F1 approximate test was significant, suggesting that the dose-response was not
monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead. Where there
were only two groups, comparisons were made using t-tests.

For litter size and survival indices and sexual maturation data: Fisher's Exact test (pairwise with each group).

Sex ratio by Wald Chi-quare test.

Gestation length, sperm count, copulation plug: exact 2-tailed linear-by-linear test. If significant, higheest dose group excluded and test reapplied (step-down repeated until significance was lost).

Estrous cycles: 1-tailed linear-by-linear test, using step down approach when significant.

Reproductive indices:

Mating performance and fertility
Individual data was tabulated. Group values were calculated for males and females
separately for the following:
Percentage mating = Number animals mating Animals paired x 100
Conception rate (%) = Number aniAmnailms aalcsh mieavtiendg pregnancy x 100
Fertility index (%) = Number anAimnaimls aalcs hpiaeivriinngg pregnancy x 100
Huntingdon Life Sciences
HJL0156
39
Gestation length
Gestation length was calculated as the number of gestation days up to and including the day
on which offspring were first observed, with Day 1 = day of mating for calculation purposes.
Where parturition had started overnight, this value was adjusted by subtracting half of one
day. Gestation index was calculated for each group as:
Gestation index (%) = Number of live litters born Number pregnant x 100

Offspring viability indices:

Survival indices
The following were calculated for each litter:
Post-implantation survival index (%) =
Total number of offspring born
Total number of uterine implantation sites x 100
Post-implantation survival index was expressed as 100% where the number of offspring
exceeded the number of implantation sites recorded.
Live birth index (%) = Number of live offspring on Day 1 after littering Total number of offspring born x 100
Viability index (%) = Number of live offspring on Day 4 before culling Number live offspring on Day 1 after littering x 100
Lactation index (%) = Number of live offspring on Day 21 after littering Number live offspring on Day 4 (after culling) x 100
Group mean values were calculated from 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 culling) and 21 of age.
Percentage males =
Number of males in litter
Total number of offspring in litter x 100
Group mean values were calculated from individual litter values.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Dose descriptor:

NOAEL

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

relative kidney weights for m/f at high dose marginally but significantly higher

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Bodyweight
Bodyweight and bodyweight gain prior to pairing for F1 males and females was not adversely
affected by treatment with [study substance] when compared with Controls.
Among males receiving 12000 ppm mean bodyweight gain during Week 4-5, 7-8 and 9-10
and during Week 7-8 for males receiving 3500 ppm was marginally but statistically
significantly low when compare with Controls.
Bodyweight gain and overall group mean bodyweight gain for F1 females prior to pairing
was unaffected by treatment. However, bodyweight gain during Weeks 9-10 prior to pairing
was statistically significantly lower for F1 females receiving MCP2484, when compared to
Controls.
At 12000 ppm, mean bodyweight gain during Days 0-20 of gestation was marginally but
statistically significantly lower than in Controls; mean bodyweights were significantly lower
than Controls from Day 14 of gestation until Day 14 of lactation. Bodyweight gain during
Days 1-14 of lactation was similar to Controls. Between Days 14 and 28 of lactation, dams
are expected to show mean bodyweight loss so that overall mean weight loss is apparent
during lactation; the extent of the overall mean weight loss in the 3500 or 12000 ppm groups
was significantly lower than in Controls.


The mean ovarian primordial follicle count in the 12000 ppm group was marginally but
statistically significantly lower than in Controls. However, no effect of treatment was
inferred since mean counts were highly variable in each group and review of the individual
values did not reveal any evidence for a treatment related trend to lower follicle counts; two
control females had atypically high mean follicle counts in excess of 32.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

sexual maturation

other: survival, growth and general condition in F1 offspring; reproductive performance in F1 adults

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: survival, growth and general condition

Reproductive effects observed:

not specified

Executive summary:

There were no clinical signs observed during the study that were related to treatment with

Dietary administration of [study substance], a base oil, to adult male and female F0 and

F1 Crl:CD (SD) rats at dietary levels up to 12000 ppm was not associated with any overt

signs of systemic toxicity, or any effects on reproductive performance.

There were no clinical signs observed during the study that were related to treatment with

[study substance].

One F0 female receiving 12000 ppm appeared to complete parturition but showed poor

clinical condition and was killed for welfare reason on Day 1 of lactation. Microscopic

examination of the uterus revealed lesions which were considered to have contributed to the

poor condition of this female and macroscopic examination revealed that parturition had been

incomplete and 5 fetuses were present in utero. Since this was a single incidence of

incomplete parturition among F0 females which was not replicated among F1 females

receiving 12000 ppm this isolated finding was considered to be unrelated to treatment.

There were no effects of treatment on oestrous cycles, pre-coital interval, mating

performance, fertility, gestation lengths and gestation index, offspring organ weights, sperm

motility, concentrations or morphology, for either generation at dietary concentrations up to

12000 ppm of [study substance]. Two F0 females receiving 12000 ppm were observed with total

litter loss, which subsequently resulted in the viability index being marginally lower at this

dose level. This was not considered to be treatment related as it was not replicated in the

F1-F2 generation; where F1 animals have greater exposure to [study substance] compared to that of the F0 generation. F1 parental animals eat treated diet from a younger age and thus through

more stages of life cycle than F0 parent animals; so if this finding was treatment related, total

litter loss should also have been apparent in F2 litters. There was no effect of treatment on

the ability of the F0 or F1 females to successfully litter and rear their offspring to weaning.

Litter size, survival of the offspring and the offspring sex ratio were not affected by treatment

of either generation.

Bodyweight, bodyweight gain and food consumption for F0 adults were unaffected by

treatment with [study substance]. Among F1 males receiving 12000 ppm mean bodyweight gain

during Week 4-5, 7-8 and 9-10 and during Week 7-8 for males receiving 3500 ppm was

marginally but statistically significantly low when compare with Controls. Bodyweight gain

during Weeks 9-10 prior to pairing was statistically significantly lower for F1 females

receiving [study substance], when compared to Controls. However, this had no impact on overall

group mean bodyweight gain prior to pairing or group mean bodyweights. At 12000 ppm,

mean bodyweight gain during Days 0-20 of gestation was marginally but statistically

significantly lower than in Controls; mean bodyweights were significantly lower than

Controls from Day 14 of gestation until Day 14 of lactation, which correlates with the

reduced food consumption at this period; the extent of the overall mean weight loss in the

3500 or 12000 ppm groups was significantly lower than in Controls. These effects on

bodyweight and food consumption were not consistent and not considered adverse as they

had no overall effect on clinical condition, survival or reproductive performance..

One F0 female receiving 12000 ppm appeared to complete parturition but showed poor

clinical condition and was killed for welfare reason on Day 1 of lactation. Microscopic

examination of the uterus revealed lesions which were considered to have contributed to the

poor condition of this female and macroscopic examination revealed that parturition had been

incomplete and 5 fetuses were present in utero. Since this was a single incidence of

incomplete parturition among F0 females which was not replicated among F1 females

receiving 12000 ppm this isolated finding was considered to be unrelated to treatment.

There were no effects of treatment on oestrous cycles, pre-coital interval, mating

performance, fertility, gestation lengths and gestation index, offspring organ weights, sperm

motility, concentrations or morphology, for either generation at dietary concentrations up to

12000 ppm of [study substance]. Two F0 females receiving 12000 ppm were observed with total

litter loss, which subsequently resulted in the viability index being marginally lower at this

dose level. This was not considered to be treatment related as it was not replicated in the

F1-F2 generation; where F1 animals have greater exposure to [study substance] compared to that of the F0 generation. F1 parental animals eat treated diet from a younger age and thus through

more stages of life cycle than F0 parent animals; so if this finding was treatment related, total

litter loss should also have been apparent in F2 litters. There was no effect of treatment on

the ability of the F0 or F1 females to successfully litter and rear their offspring to weaning.

Litter size, survival of the offspring and the offspring sex ratio were not affected by treatment

of either generation.

Bodyweight, bodyweight gain and food consumption for F0 adults were unaffected by

treatment with [study substance]. Among F1males receiving 12000 ppm mean bodyweight gain

during Week 4-5, 7-8 and 9-10 and during Week 7-8 for males receiving 3500 ppm was

marginally but statistically significantly low when compare with Controls. Bodyweight gain

during Weeks 9-10 prior to pairing was statistically significantly lower for F1 females

receiving [study substance], when compared to Controls. However, this had no impact on overall

group mean bodyweight gain prior to pairing or group mean bodyweights. At 12000 ppm,

mean bodyweight gain during Days 0-20 of gestation was marginally but statistically

significantly lower than in Controls; mean bodyweights were significantly lower than

Controls from Day 14 of gestation until Day 14 of lactation, which correlates with the

reduced food consumption at this period; the extent of the overall mean weight loss in the

3500 or 12000 ppm groups was significantly lower than in Controls. These effects on

bodyweight and food consumption were not consistent and not considered adverse as they

had no overall effect on clinical condition, survival or reproductive performance.

Among F2 offspring in the 12000 ppm group, mean body weight gains between Days 1 and

21 were marginally (less than 10%) lower than in Controls. However, this minor effect of

treatment on the growth of the offspring is considered not to be adverse since there was no

effect on the survival or general condition of the offspring.

Absolute and bodyweight relative pituitary weights for F1 adult males receiving 12000 ppm

were marginally but statistically significantly lower than in Controls and bodyweight relative

kidney weights for F1 adult males and females receiving 12000 ppm were marginally but

significantly higher than in Controls. These findings were considered not to be of any

toxicological significance as there were no corresponding macroscopic or histopathological

findings.

Macroscopic and histopathological examination of the F0 and F1 adult males and females did

not reveal any findings that were considered to be related to treatment with [study substance].

Macroscopic examination of any abnormal tissues of F1 and F2 offspring did not reveal any

findings that were related to treatment with [study substance] at dietary levels up to 12000 ppm.

The mean ovarian primordial follicle count in the 12000 ppm group was marginally but

statistically significantly lower than in Controls. However, no effect of treatment was

inferred since mean counts were highly variable in each group and review of the individual

values did not reveal any evidence for a treatment related trend to lower follicle counts;

2 control females had atypically high mean follicle counts in excess of 32. The mean number

of implantation in these females was identical to Controls so even if the marginally lower

primordial follicle counts were viewed as treatment related it would not be viewed as an

adverse finding within the context of this study as it did not affect reproductive performance.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The potential for adverse effects on reproduction following exposure to the study substance was evaluated by read across to data available with reaction products of 1 -tetradecene and naphthalene according to the methods described in OECD TGs 421 and 416.

In the OECD 421 study, The test material, administered in the diet had no clear adverse effects upon male or female rats, but

was associated with slightly increased weight gain in the FO females at the highest level (12000ppm). A close inspection of the exact timing of the oestrous cycles showed that the majority of high dose females were cycling synchronously such that 9/10 females were in

di-oestrus on the final day of the pre-pairing period. The female rat normally loses a small amount of bodyweight over the night between protesters and oestrus. The absence of any rats in oestrus on the final weigh day and the presence of two animals which had gone acyclic

may make a significant difference to the overall weight gain of these animals at this point. These changes are considered to be part of the normal physiological pattern and not to represent any adverse effect upon either the cycles or the female weight gain patterns.

It is concluded that dietary administration of the test material (a base oil), at concentrations up to 12000 ppm caused no adverse effect on reproductive performance and development in the rat. The no-observed- adverse-effect level (NOAEL) in this study was 12000 ppm, which is approxumately equivalent to an actual dose of 1000 mg/kg/day, which is the generally accepted maximum desirable exposure level for studies of this type.

In the OECD 416 study, There were no clinical signs observed during the study that were related to treatment with

Dietary administration of [test substance], a base oil, to adult male and female F0 and F1 Crl:CD (SD) rats at dietary levels up to 12000 ppm was not associated with any overt signs of systemic toxicity, or any effects on reproductive performance. There were no clinical signs observed during the study that were related to treatment with [test substance].

One F0 female receiving 12000 ppm appeared to complete parturition but showed poor clinical condition and was killed for welfare reason on Day 1 of lactation. Microscopic examination of the uterus revealed lesions which were considered to have contributed to the poor condition of this female and macroscopic examination revealed that parturition had been incomplete and 5 fetuses were present in utero. Since this was a single incidence of incomplete parturition among F0 females which was not replicated among F1 females receiving 12000 ppm this isolated finding was considered to be unrelated to treatment.

There were no effects of treatment on oestrous cycles, pre-coital interval, mating performance, fertility, gestation lengths and gestation index, offspring organ weights, sperm motility, concentrations or morphology, for either generation at dietary concentrations up to

12000 ppm of [test substance].

Two F0 females receiving 12000 ppm were observed with total litter loss, which subsequently resulted in the viability index being marginally lower at this dose level. This was not considered to be treatment related as it was not replicated in the F1-F2 generation; where F1 animals have greater exposure to [test substance] compared to that of the F0 generation. F1 parental animals eat treated diet from a younger age and thus through more stages of life cycle than F0 parent animals; so if this finding was treatment related, total

litter loss should also have been apparent in F2 litters. There was no effect of treatment on the ability of the F0 or F1 females to successfully litter and rear their offspring to weaning. Litter size, survival of the offspring and the offspring sex ratio were not affected by treatment of either generation.

Bodyweight, bodyweight gain and food consumption for F0 adults were unaffected by treatment with [test substance]. Among F1males receiving 12000 ppm mean bodyweight gain during Week 4-5, 7-8 and 9-10 and during Week 7-8 for males receiving 3500 ppm was marginally but statistically significantly low when compare with Controls. Bodyweight gain during Weeks 9-10 prior to pairing was statistically significantly lower for F1 females receiving [test substance], when compared to Controls. However, this had no impact on overall group mean bodyweight gain prior to pairing or group mean bodyweights. At 12000 ppm, mean bodyweight gain during Days 0-20 of gestation was marginally but statistically significantly lower than in Controls; mean bodyweights were significantly lower than Controls from Day 14 of gestation until Day 14 of lactation, which correlates with the reduced food consumption at this period; the extent of the overall mean weight loss in the 3500 or 12000 ppm groups was significantly lower than in Controls. These effects on bodyweight and food consumption were not consistent and not considered adverse as they had no overall effect on clinical condition, survival or reproductive performance..

Among F2 offspring in the 12000 ppm group, mean body weight gains between Days 1 and

21 were marginally (less than 10%) lower than in Controls. However, this minor effect of

treatment on the growth of the offspring is considered not to be adverse since there was no

effect on the survival or general condition of the offspring.

Absolute and bodyweight relative pituitary weights for F1 adult males receiving 12000 ppm

were marginally but statistically significantly lower than in Controls and bodyweight relative

kidney weights for F1 adult males and females receiving 12000 ppm were marginally but

significantly higher than in Controls. These findings were considered not to be of any

toxicological significance as there were no corresponding macroscopic or histopathological

findings.

Macroscopic and histopathological examination of the F0 and F1 adult males and females did

not reveal any findings that were considered to be related to treatment with [test substance].

Macroscopic examination of any abnormal tissues of F1 and F2 offspring did not reveal any

findings that were related to treatment with [test substance] at dietary levels up to 12000 ppm.

The mean ovarian primordial follicle count in the 12000 ppm group was marginally but

statistically significantly lower than in Controls. However, no effect of treatment was

inferred since mean counts were highly variable in each group and review of the individual

values did not reveal any evidence for a treatment related trend to lower follicle counts;

2 control females had atypically high mean follicle counts in excess of 32. The mean number

of implantation in these females was identical to Controls so even if the marginally lower

primordial follicle counts were viewed as treatment related it would not be viewed as an

adverse finding within the context of this study as it did not affect reproductive performance.

Additionally, a two generation reproductive toxicity study (OECD 416) in which the structurally analogous diisopropylnaphthalene was administered by oral gavage during gestation reported no effects on any of the reproductive parameters examined including pregnancy rate, litter size and survival rates (ECB, 2000).

Taken together, these data are adequate to support the conclusion that the study substance does not present a reproductive toxicity hazard.

Short description of key information:

No key studies available. No testing required based on weight of evidence.

Effects on developmental toxicity

Description of key information

Prenatal developmental toxicity study, 1 key study, NOAEL= 1000 mg/kg/day for rat (OECD TG 414).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report according to OECD guideline 414: GLP .

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

Identification: MCP2484
Alternative name: SYNESSTIC 12
Action: Base oil
Description: Amber liquid
Storage conditions: At ambient temperature
Supplier: Sponsor
Batch number: E09K005
Date of receipt: 7 June 2010
Quantity received: 3 x 1L
Expiry date: 31 December 2011

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Females were treated from Day 6 to Day 19 after mating. Animals received the test material or vehicle control formulations orally at a volume-dose of 4 mL/kg bodyweight, using a suitably graduated syringe and a rubber catheter inserted via the mouth into the stomach.
All animals were dosed in sequence of cage-number within each group, once each day at approximately the same time as the previous day, seven days per week. The volume administered to each animal was calculated from the most recently recorded bodyweight. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of the test material in the liquid matrix was demonstrated as 15 days at 2-8°C (refrigerated) and 24 hours when stored at ambient temperature. During the first and last weeks of treatment, the test formulations were analysed for achieved concentration of the test substance. Four samples (nominally 1 mL accurately weighed) were taken from all groups. Two assays from each group were analysed and the remainder were retained refrigerated (nominally 2-8 ºC) as contingency.

Details on mating procedure:

Crl: CD (SD) rats (a total of 90 females) were received from Charles River (UK) Ltd. They were ordered at approximately 65 days of age and within a weight range of 227 to 254g. On receipt animals appeared healthy. Within 24 hours of arrival, a representative sample of 15 animals was weighed and these animals were within the weight range. The animals were checked twice daily for health and general condition and were allowed to acclimatise to the conditions described below for five days before they were paired on a 1:1 basis with stock males from the same source. Daily checks were made after pairing for evidence of mating, including ejected copulation plugs in cage trays and the presence of sperm in a vaginal smear. Animals were allocated to study on Day 0 of gestation, when positive evidence of mating was detected. Only females with a sperm positive vaginal smear or at least two copulation plugs were selected. Females were allocated to group and cage position in sequence of mating, thus ensuring that animals mated on any one day were evenly distributed amongst the groups. The sequence of allocation was controlled to prevent stock males from providing more than one mated female in each treatment group.

Duration of treatment / exposure:

Females were treated from Day 6 to Day 19 (inclusive) after mating

Frequency of treatment:

All animals were dosed in once each day at approximately the same time as the previous day, seven days per week.

Duration of test:

Study initiation:
(Protocol signed by Study Director)
3 September 2010
Experimental start date:
(Animal arrival)
22 September 2010
Pairing commenced: 27 September 2010
Treatment commenced: 4 October 2010
Necropsy completed: 21 October 2010
Experimental completion date:
01 December 2010
Study completion: 20 January 2011

No. of animals per sex per dose:

20 females/dose

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult female was recorded on Days 0, 3 and 6-20 after mating.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- The weight of food supplied to each adult female, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. After ventral midline incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. External and cut surfaces of the organs and tissues were examined as appropriate. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

Ovaries and uterine content:

The following reproductive assessment was made for all animals after termination:
The gravid uterus was weighed before removal of the fetuses; this weight included the weight of the cervix, uterus, fallopian tubes and ovaries.
For each animal, the number of corpora lutea in each ovary and the number of implantation sites, the number and distribution of resorption sites (classified as early or late) and live and dead fetuses were recorded for each uterine horn. The retained tissues were checked before disposal of the carcass.

Fetal examinations:

All fetuses and placentae were dissected from the uterus and weighed individually. Fetuses were individually identified within the litter, using a coding system based on their position in the uterus. Each fetus and placenta was externally examined and any abnormalities were recorded. The sex of each fetus was recorded. Approximately half of the fetuses in each litter were eviscerated and their skeletons were fixed in Industrial Methylated Spirit, prior to processing and staining with Alizarin Red and Alcian Blue. The remaining fetuses were fixed whole in Bouin’s fluid. Free-hand serial sections were prepared from the Bouin’s fixed fetuses and were examined under the microscope for visceral abnormalities. Fetuses stained with Alizarin Red and Alcian Blue were assessed for skeletal and cartilage development and abnormalities.

Findings observed were classified, according to severity and incidence, as:
Major abnormalities: normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. ventricular septal defect
Minor abnormalities: minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated urether.
Variants: alternative structures or stages of development occuring regularly in the control population, e.g. number of ribs, incomplete ossification of 5th and 6th sternebrae.
In the Liberate Fetal Pathology Appendix, observations on repeated structures like ribs, vertebrae and sternebrae are reported as the first and last affected element, in the form “5th 13th bilateral ribs”, which should be interpreted as “5th to 13th bilateral ribs

Statistics:

Statistical analyses were applied where there was indication of possible meaningful intergroup differences. All statistical analyses were carried out using the individual animal (or litter) 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.
The following data types were analysed at each timepoint separately:
Bodyweight, using absolute weights and gains over appropriate study periods;
Gravid uterine weight, adjusted bodyweight and bodyweight change;
Food consumption, using means over appropriate study periods;
Litter size and survival indices;
Fetal, placental and litter weight.

The following sequence of statistical tests was used for bodyweight, food consumption, litter size and survival indices and fetal placental and litter weight data: A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose-response was not monotone, Dunnett's test was performed instead. A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations.. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed.

Indices:

Individual values are presented for the numbers of corpora lutea, implantations, resorptions (early, late and total) and live fetuses (male, female, total) and sex ratio (percentage male) for litters at Day 20 of gestation. Prenatal losses are separated into pre- and post implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilisation of ova but may include very early post-implantation deaths (i.e. those occurring during the first two to three days post-implantation), in addition to true pre-implantation loss. It was calculated from the formula:
(Number of corpora lutea – Number of Pre-implantation loss (%) = implantations)
Number of corpora lutea
x 100
Where the number of implantations exceeded the number of corpora lutea observed,
pre-implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to
have occurred).
Post-implantation loss was considered to exclude the first two to three days post-implantation
as deaths occurring at this stage are considered to leave no remains visible at Day 20 of
gestation. It was calculated from the formula:
Post-implantation loss (%) = (Number of implantations – Number of live fetuses) Number of implantations x 100
All group values and SD (as appropriate) were calculated from the individual litter values.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Maternal clinical condition, bodyweight, food consumption and macroscopic evaluation was unaffected by treatment with MCP2484 upto 1000 mg/kg/day when compared with Control animals.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Embryo fetal survival growth and external morphology were unaffected by treatment with MCP2484 upto 1000 mg/kg/day.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: developmental toxicity, embryo fetal survival

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Oral administration of MCP 2484, (a base oil), to Crl:CD(SD) rats during the organogenesis phase of embryo fetal development resulted no adverse effects being observed at dose levels upto 1000 mg/kg/day. It was concluded from this study that the dosage of 1000 mg/kg/day was the maternal noobserved- adverse-effect-level (NOAEL) and 1000 mg/kg/day was the no-observed-adverseeffect- level (NOAEL) for embryo fetal survival and development. These findings do not warrant classification of the test material for developmental/reproductive toxicity under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP), under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations, or under the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

Executive summary:

The influence of MCP2484 (a base oil) on embryo-fetal survival and development in Crl:CD(SD) rats was assessed following oral administration during and after the organogenesis phase of pregnancy (Days 6-19 after mating). Three groups of twenty female rats received MCP2484 by gavage at doses of 100, 300 or 1000 mg/kg/day from Day 6 to 19 after mating, inclusive. A similarly constituted Control group received the vehicle, corn oil, at the same dose volume throughout the same period. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination. During the study, clinical condition, dosing signs, bodyweight and food consumption were monitored. Adult females were examined macroscopically at necropsy on Day 20 after mating and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal and cartilaginous examination.

Maternal clinical condition, bodyweight, food consumption and macroscopic evaluation was unaffected by treatment with MCP2484 upto 1000 mg/kg/day when compared with Control animals. Embryo fetal survival growth and external morphology were unaffected by treatment with MCP2484 upto 1000 mg/kg/day.

It was concluded from this study that the dosage of 1000 mg/kg/day was the maternal no observed- adverse-effect-level (NOAEL) and 1000 mg/kg/day was the no-observed-adverse effect- level (NOAEL) for embryo fetal survival and development

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

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

The potential for adverse effects on reproduction and in utero development following exposure to the study substance was evaluated by read across to data available with reaction products of 1 -tetradecene and naphthalene according to the methods described in OECD guideline 414. In this study, three groups of twenty female rats received test article by gavage at doses of 100, 300 or 1000 mg/kg/day from Day 6 to 19 after mating. A similarly constituted control group received the vehicle, corn oil, at the same dose volume throughout the same period. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination. Maternal clinical condition, bodyweight, food consumption and macroscopic evaluation were unaffected by treatment with test material up to 1000 mg/kg/day when compared with controlanimals. Embryo fetal survival, growth, macroscopic pathology, and external morphology (both internal visceral examination and skeletal cartilaginous examination) were unaffected by treatment with test material up to 1000 mg/kg/day. It was concluded from this study that the dosage of 1000 mg/kg/day was the maternal no observed- adverse-effect-level (NOAEL) and 1000 mg/kg/day was the no-observed-adverse effect- level (NOAEL) for embryo fetal survival and development.

Additionally, toxicokinetic data and physico-chemical properties support limited absorption of the study substance by all routes of exposure..  Second, three separate studies covering two different species on a structurally analogous alkylated naphthalene material, diisopropylnaphthalene, failed to demonstrate teratogenic effects at doses that were not maternally toxic (EPA, 2003; LPT, 1993; ECB, 2000). DIPN is an adequate substance for use as read across based on similar functionality and metabolism as the study substance (refer to the toxicokinetic section for more detail). In these repeated-dose developmental toxicity studies, no effects were observed on survival rates, fetal body weight, newborn growth data, litter size, or on variation and/or developmental retardations of soft tissue. Small effects noted at maternally toxic doses included decreased fetal body weight in one study (EPA, 2003) and possible (not statistically significant) treatment-related skeletal retardations in two studies (EPA, 2003; LPT, 1993). However, because these observations were noted only at dose levels inducing maternal toxicity they cannot be considered as specific effects on prenatal development. 

These data are adequate to conclude the study substance has a low potential for developmental toxicity.

Justification for classification or non-classification

Based on assessment of the available data, there is no evidence of reproductive toxicity warranting classification under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP), under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations, or under the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).