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Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 November 2011 - 23 March 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
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:
TEST ANIMALS
- Source: breeder: Charles River Laboratories France, L’Arbresle, France
- Age/Weight: at the beginning of the treatment period, the males were approximately 10 weeks old and weighed approximately 384 g (range 358 g to 414 g). The females were approximately 9 weeks old and weighed approximately 212 g (range 185 g to 231 g)
- Fasting period before study: no
- Housing: The animals were individually housed, except during pairing, in polycarbonate cages with stainless steel lids and containing autoclaved sawdust. Toward the end of gestation and during lactation with their litter, autoclaved wood shavings
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00).

IN-LIFE DATES: 22 November 2011 to 15 January 2012
Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous methylcellulose solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder, using a mortar and pestle, and then mixed with the required quantity of vehicle.
The test item dosage forms were prepared daily in the vehicle.
The dosage forms were delivered to the study room at room temperature.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurs or 14 days has elapsed
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred to as day 0 post-coitum
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: HPLC-UV,
The concentration of the lactame content in each control and test item dosage form prepared for use in weeks 1, 3 and 5 were determined (three occasions). Concentration of test item in the dosage forms were then determined accordingly.
The dosage form preparations were delivered to the study room before acceptance of the results of the concentration.

Homogeneity: the dosage forms containing the test item in 0.5% methylcellulose in drinking water treated by reversed osmosis at 5 and 200 mg/mL were found to be homogeneous.
Stability: not assessed, dose formulation prepared daily
Duration of treatment / exposure:
The dosage forms were administered daily according to the following schedule:
in the males:
- 2 weeks before pairing,
- during the pairing period (3 weeks),
- until sacrifice (at least 5 weeks in total),

in the females:
- 2 weeks before pairing,
- during the pairing period (3 weeks),
- during gestation,
- during lactation until day 5 p.p. inclusive,
- until sacrifice for females with no delivery.

Day 1 corresponds to the first day of the treatment period.
Frequency of treatment:
Once daily.
Details on study schedule:
- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: 12 weeks for males and 10 weeks for females.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, following the results of a previous studies.

- Rationale for animal assignment: computerized stratification procedure.
Positive control:
no (not required)
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

BODY WEIGHT (GAIN):
- Time schedule: The body weight of each male was recorded on the first day of treatment (day 1), then once a week until sacrifice.
The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mating or until sacrifice and on days 0, 7, 14 and 20 post-coitum and on days 1 and 5 p.p..

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by each male was measured once a week, over a 7 day period, from the first day of treatment until the start of the pairing period.
The quantity of food consumed by each female was measured once a week, over a 7 day period, from the first day of treatment until the start of the pairing period, during pregnancy at the intervals days 0-7, 7-14 and 14-20 p.c. and during lactation for interval day 1 5 p.p.
During the pairing period, the food consumption was measured for neither males nor females.
Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.

NEUROBEHAVIOURAL EXAMINATION:
The first five males and the first five females to deliver from each group were evaluated once at the end of the treatment period. For the males, this was conducted after about 5 weeks of treatment and for females, on day 5 p.p. after sacrifice of the pups.

HAEMATOLOGY:
- Time schedule: on the day of sacrifice.

CLINICAL CHEMISTRY:
- Time schedule: on the day of sacrifice.

URINALYSIS:
- Time schedule: on the day of sacrifice.

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded.
Oestrous cyclicity (parental animals):
fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until females are mated.
Sperm parameters (parental animals):
Parameters examined in males of parental generation:
- weighing and microscopic examination: see Tissue Procedure Table below
- microscopic evaluation of stages of the spermatogenic cycle and testicular interstitial cells (HD group)
Litter observations:
STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs

GROSS EXAMINATION OF DEAD AND SURVIVING PUPS:
- external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals after the end of the mating period
- Female animals: all surviving animals = day 4 post-partum or, for females which had not delivered yet, day 25 post-coitum (mothers with litter dying entirely were sacrificed as appropriate)

ORGAN WEIGHTS: see table below

GROSS PATHOLOGY:
Complete macroscopic post-mortem examination

HISTOPATHOLOGY:
- on all tissues listed in the table below for the first five control and high dose animals (groups 1 and 4) sacrificed as scheduled,
- on all macroscopic lesions,
- all females sacrificed because of non-delivery to investigate possible causes.
Postmortem examinations (offspring):
SACRIFICE: on day 5 post-partum

GROSS NECROPSY: on all pups (surviving and found dead)

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No
Reproductive indices:
Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live concepti) / Number of implantations
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)
Offspring viability indices:
Live birth index = 100 * (Number of live born pups / Number of delivered pups)
Viability index on day 4 p.p. = 100 * (Number of surviving pups on day 4 p.p. / Number of live born pups)
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:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
no in-vivo observation of sperm
Reproductive performance:
no effects observed
MORTALITY:
Male
No deaths were observed prior to scheduled sacrifice.

Female
At 100 mg/kg/day: one female was prematurely sacrificed on day 23 of pregnancy due to difficult delivery. Piloerection, pallor of extremities and pallor of eyes were noted before sacrifice. At macroscopic post-mortem examination yellow discoloration of the thyroid glands, enlarged spleen, discoloration of the liver, red content in the uterus and vagina were noted. This female had a transverse vaginal septum, therefore a treatment-related effect was excluded.

At 300 mg/kg/day: one female was sacrificed on day 26 post-coitum (p.c.) due to no delivery. This female was non-pregnant.

CLINICAL SIGNS:
Male
In rats given 1000 mg/kg/day, ptyalism was generally observed from week 3 until the end of the treatment period.
In rats given 300 mg/kg/day, ptyalism was observed on day 27 only.

Female
. Mating period
In females given 1000 mg/kg/day ptyalism was observed from day 17.

. Gestation - lactation periods
In females given 1000 mg/kg/day, ptyalism was generally observed during all the gestation and lactation periods.
In females given 300 mg/kg/day, ptyalism was observed from day 10 p.c. and for up to 3 days during the lactation period.

Overall, excessive salivation (ptyalism) was observed in males treated from 300 mg/kg/day and in females at 1000 mg/kg/day. While treatment-related, ptyalism was considered to be non-adverse at 300 mg/kg/day taking into account the incidence and duration of this finding at this dose-level.

BODY WEIGHT (GAIN):
There were no dose-related effects on mean body weight and mean body weight change neither in males nor in females during the pre-mating, mating or pregnancy periods.

FOOD CONSUMPTION:
There were no effects on mean food consumption neither in males nor in females during the pre mating, mating periods or pregnancy period.

NEUROBEHAVIOURAL EXAMINATION:
There were no relevant differences in treated group animals when compared to control animals.
There was a tendency toward a decrease in rearing at 1000 mg/kg/day in males. There were no effects in females.
The relevance of this finding was not clear since no clinical signs were recorded which confirmed hypoactivity in males.
Overall, it was considered that there were no relevant differences in treated group animals when compared to control animals.

HAEMATOLOGY:
There were no dose-related differences in hematological parameters when compared with control group. Therefore a test item treatment-related effect was considered unlikely.

CLINICAL CHEMISTRY:
The only relevant findings were lower glucose and bile acid levels (statistical significant: p<0.05) at 1000 mg/kg/day in males.

Taking into account the amplitude of these changes and the absence of any effects in females, a test item treatment-related effect was considered to be unlikely.

URINALYSIS:
There were no effects on mean urinalysis parameters.

REPRODUCTIVE PERFORMANCE:
There were no effects on mating and fertility performance in treated animals when compared with controls.
There were no effects on the mean numbers of corpora lutea, implantations or pups at any dose level, nor on the duration of gestation or the extent of pre- and post-implantation losses.

ORGAN WEIGHTS:
Higher relative liver weights were noted in females treated at 1000 mg/kg/day. This weight difference correlated with hepatocellular hypertrophy and was considered to be treatment-related.
Other weight differences were minimal, or of opposing trend between the groups or sexes. Therefore a relationship to treatment was excluded.

GROSS PATHOLOGY:
Premature deaths
Female treated at 100 mg/kg/day was euthanized on day 39 on ethical grounds. A transverse vaginal septum was noted, along with several dead fetuses in the uterus horns. This finding is a cause of dystocia and was considered to be a major contributing factor to death.

Terminal sacrifice
There were no treatment-related macroscopic findings.

The gross findings observed were of those commonly reported in the rat of this strain and age and a relationship to treatment was considered to be unlikely.

HISTOPATHOLOGY:
Premature deaths
Female X21461 treated at 100 mg/kg/day was euthanized on day 39 on ethical grounds. No microscopic findings were attributed to treatment.

Terminal sacrifice
Microscopic findings were observed in the mesenteric lymph nodes, in the liver and in the kidneys as follows.

Mesenteric lymph nodes
Macrophages aggregates were observed in the mesenteric lymph nodes, along with lymphoid hyperplasia (increased size/numbers of germinal centers and expansion of the paracortex area).
Macrophages aggregates were recorded in males from 100 mg/kg/day onward and in females from 300 mg/kg/day. These findings suggested inflammation and reactive lymphoid hyperplasia, possibly as a result of test item draining from the digestive tract.

Liver
Minimal to slight hepatocellular hypertrophy was noted in the liver in five out of five males examined treated at 1000 mg/kg/day and in females from 100 mg/kg/day onwards. As this finding was not associated with any hepatocellular degeneration/necrosis, this hypertrophy was considered not to be adverse.

Kidneys
Hyaline tubular droplets were seen in the kidneys in one out of six males treated at 300 mg/kg/day and in the six males examined that were treated at 1000 mg/kg/day.
As there were no biochemical or hematological changes, this finding was considered not to be adverse.

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
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
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
MORTALITY/VIABILITY:
When compared with the control group, there were no effect of the number of pups found dead, prematurely sacrificed or cannibalized and the number of litters affected.

CLINICAL SIGNS:
When compared with the control group, there were no effect of the number of pups found dead, prematurely sacrificed or cannibalized and the number of litters affected.

BODY WEIGHT (GAIN):
There were no effects on mean body weight neither in male nor in female pups during the lactation period (day 1 or day 5 p.p.).

GROSS PATHOLOGY:
There were no treatment-related findings on macroscopic examination of the principal thoracic and abdominal organs of pups sacrificed on day 5 p.p.
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Reproductive effects observed:
no

Mating data are summarizedas follows:

 

Dose-level(mg/kg/day)

0

100

300

1000

Number of animals paired(M +F)

10 +10

10 +10

10 +10

10 +10

Number of males mated

10(a)

10

10

10

Number of females mated

10(a)

10

10

10

Mean number of days taken to mate

2.7

2.5

2.4

3.6

Number of pregnant females

10

10

9

10

Male fertility index (%)

100

100

100

100

Female fertility index (%)

100

100

90

100

Female gestation index (%)

100

90

100

100

(a):one female with no evidence of mating (no spermatozoa in the vaginals mear) but pregnant,was mated with its first partner.

Conclusions:
The test item, Dodecane-12-Lactam Manufacturing of, by-products from, distillation residues, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and (for females) throughout gestation and until day 5 post partum, at dose-levels of 100, 300 or 1000 mg/kg/day.

In parent animals, there were effects in the liver, mesenteric lymph nodes and in the kidneys.
- in pups, there were no treatment-related findings.

Based on the experimental conditions of this study:
- the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day,
- the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day in the absence of significant effects on mating and fertility at this dose-level,
- the No Observed Effect Level (NOEL) for toxic effects on progeny was considered to be 1000 mg/kg/day in the absence of any treatment-related effect on pups at this dose-level.
Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item, Dodecane-12-Lactam Manufacturing of, by-products from, distillation residues, following daily oral administration (gavage) to male and female rats from before mating, through mating and, for the females, through gestation until day 5 post-partum (p.p.) inclusive.

.

Three groups of ten male and ten female Sprague-Dawley rats received the test item, Dodecane-12-Lactam Manufacturing of, by-products from, distillation residues, daily, by oral (gavage) administration, before mating, during mating and, for the females, throughout gestation until day 5 p.p., at dose-levels of 100, 300 or 1000 mg/kg/day. An additional group of ten males and ten females received the vehicle control, 0.5% methylcellulose aqueous solution, under the same experimental conditions.

 

Animals were checked daily for clinical signs, mortality, and detailed clinical observations were conducted weekly. Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation.

The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5 p.p.. The total litter sizes and numbers of pups of each sex were recorded after birth. The pups were observed daily for clinical signs of toxicity and pup body weights were recorded on days 1 and 5 p.p.

Dams were sacrificed on day 6 p.p. Body weights and selected organs weights were recorded and a complete macroscopic examination was performed, with particular attention paid to the reproductive organs. A microscopic examination was then conducted on selected organs from the first five females to deliver in the control group and the high-dose group and on any macroscopic lesions from all groups.

Based upon the microscopic results of the high-dose group, liver and spleen tissues of the first five animals of the low- and intermediate-dose groups were examined.

 

Pups, including those found dead before study termination, were also submitted for a macroscopic post-mortem examination.

There were no treatment-related deaths. Overall, excessive salivation (ptyalism) was observed in males treated from 300 mg/kg/day and in females at 1000 mg/kg/day. While treatment-related, ptyalism was considered to be non-adverse at 300 mg/kg/day taking into account the incidence and duration of this finding at this dose-level.

There were no treatment-related effects on mean body weight and mean body weight change and food consumption neither in males nor in females.

There were no significant toxicological effects on mean hematology , blood biochemistry or urinlaysis parameters.

 

Pairing, mating and fertility

There were no treatment-related effects on pairing, mating and fertility parameters.

 

Pup observations

There were no effects on live birth, viability and lactation indexes. There were no treatment related findings in pups at necropsy.

 

Pathology

Higher relative liver weights were noted in females treated at 1000 mg/kg/day. This weight difference correlated with hepatocellular hypertrophy and was considered to be treatment-related.

There were no treatment-related macroscopic findings.

Microscopic findings were observed in the mesenteric lymph nodes, in the liver and in the kidneys as follows: macrophages aggregates were observed in the mesenteric lymph nodes, most of the time with lymphoid hyperplasia (increased size/numbers of germinal centers and expansion of the paracortex area) from 100 mg/kg/day in males and from 300 mg/kg/day in females. Minimal to slight hepatocellular hypertrophy was noted in the liver in males treated at 1000 mg/kg/day and in females from 100 mg/kg/day onwards. Hyaline tubular droplets were seen in the kidneys in a single male treated at 300 mg/kg/day and in all males examined at 1000 mg/kg/day.

These findings were considered not to be adverse.


Conclusion

The test item, Dodecane-12-Lactam Manufacturing of, by-products from, distillation residues,was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and (for females) throughout gestation and until day 5 post-partum, at dose-levels of 100, 300 or 1000 mg/kg/day.

 

In parent animals, there were effects in the liver, mesenteric lymph nodes and in the kidneys that were not considered as adverse effects.

.         in pups, there were no treatment-related findings.

 

Based on the experimental conditions of this study:

.         the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day,

.         the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day in the absence of significant effects on mating and fertility at this dose-level,

.         the No Observed Effect Level (NOEL) for toxic effects on progeny was considered to be 1000 mg/kg/day in the absence of any treatment-related effect on pups at this dose-level.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

The test item, Dodecane-12-Lactam Manufacturing of, by-products from, distillation residues,was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and (for females) throughout gestation and until day 5post-partum, at dose-levels of 100, 300 or 1000 mg/kg/day.

 

In parent animals, there were effects in the liver, mesenteric lymph nodes and in the kidneys that were not considered as adverse effects. There were no effects on fertility, mating, pregnancy delivery or prenatal development of the pups.

 

Based on the experimental conditions of this study:

. The No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day,

.  The No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day in the absence of significant effects on mating and fertility at this dose-level,

.   The No Observed Effect Level (NOEL) for toxic effects on progeny was considered to be 1000 mg/kg/day in the absence of any treatment-related effect on pups at this dose-level.


Short description of key information:
There was one study perfomed according to GLP and OECD 422 guideline. This study provides initial information on possible health hazards (including neurological and immunological effects) likely to arise from repeated exposure over a limited period of time. It can also indicate effects on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.No effects were observed on fertility or prenatal parameters at dose-levels of 100, 300 and 1000 mg/kg/day.
No other study was available.

Justification for selection of Effect on fertility via oral route:
The study was perfomed according to GLP and OECD 422 guideline.

Justification for classification or non-classification

Additional information

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