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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 April 2012 - 02 July 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N-HEPTANOL
- Physical state: colorless liquid
- Analytical purity: 99.81%
- Lot/batch No.: 1103007
- Expiration date of the lot/batch: 28 October 2011
- Storage conditions of test material: at room temperature in fireproof storage cabinet.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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 had a mean body weight of 407 g (range: 376 g to 435 g) and the females were approximately 9 weeks old and had a mean body weight of 220 g (range: 205 g to 234 g)
- Fasting period before study: no
- Housing: individually housed, except during pairing, in polysulfone cages with stainless steel lids
- 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 6 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.

IN-LIFE DATES: 09 May 2012 to 02 July 2012.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. The test item was mixed with the required quantity of vehicle and stirred at least 30 minutes. No correction factor was applied.
The dose formulations were prepared for 1 to 8 days (frequency based on the results of the stability study. They were stored at room temperature and protected from light and delivered to the study room in brown flasks.

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 21days 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: GC-FID
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: not assessed, dose formulation is a solution
Stability: 8-day
Duration of treatment / exposure:
 in the males:
- 2 weeks before pairing,
- during the pairing period,
- until sacrifice (at least 5 weeks in total),
 in the females:
- 2 weeks before pairing,
- during the mating period,
- during gestation,
- during lactation until day 5 p.p. inclusive,
- until sacrifice for non-pregnant females.
Frequency of treatment:
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, 11 weeks for females.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
actual ingested
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.
In a previous preliminary study, the test item was administered to three groups of three male and three female Sprague-Dawley rats by oral gavage once daily at 100, 300 or 1000 mg/kg/day (5 mL/kg/day was used). An additional group of three males and three females was administered the vehicle alone, corn oil, under the same experimental conditions (2 weeks). The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded twice weekly. On completion of the treatment period and after at least 14 hours fasting, all animals were sacrificed. A complete macroscopic post mortem examination was performed on all animals and designated organs were weighed. No microscopic examination was performed.

There were no unscheduled deaths. At 1000 mg/kg/day, all animals (male and female rats) had ptyalism. This finding was considered to be related to treatment with the test item but of minor toxicological importance. There were no toxicologically significant effects on mean body weight, mean body weight change or mean food consumption. At necropsy, no organ weight changes or gross finding were attributed to n-HEPTANOL administration.

Therefore 1000 mg/kg/day, which is also the maximum recommended dose to be tested according to OECD Guideline No. 422, was selected as the high-dose level. The low-dose and mid-dose were selected using a ratio representing a three-fold interval (i.e. 100 and 300 mg/kg/day).

- Rationale for animal assignment: computerized stratification procedure
Positive control:
no (not required)

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: at least twice a day during the treatment period.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: once a day during the treatment period.

BODY WEIGHT (GAIN):
- Time schedule: Males: on the first day of treatment, then once a week until sacrifice. Females: on the first day of treatment, then once a week until mating (or until sacrifice), on days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION:
- Time schedule: once a week until sacrifice, with the exception of the mating period (not recorded).

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 females, this was performed on day 5 post partum after sacrifice of the pups.

HAEMATOLOGY:
On the day of sacrifice.

CLINICAL CHEMISTRY:
On the day of sacrifice.

URINALYSIS:
On the day of sacrifice.

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded.
Estrous 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.
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 5 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
The body weight of each animal sacrificed as scheduled (see § Study plan adherence) was recorded before sacrifice, and the organs specified in the Tissue Procedure Table were weighed (wet) as soon as possible after dissection.
The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all parent animals including one male of group 4 which was sacrificed prematurely. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. The numbers of corpora lutea and implantation sites were also recorded for females sacrificed as scheduled on day 6 p.p., and for females sacrificed on day 25 p.c. due to no delivery.
For apparently non-pregnant females the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

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)

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
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
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: sperm measures:
not examined
Description (incidence and severity):
no in-vivo observation of sperm
Reproductive performance:
no effects observed

Details on results (P0)

MORTALITY:
There were no unscheduled deaths in control, 100 and 300 mg/kg/day groups.
At 1000 mg/kg/day, one male was sacrificed moribund (hypoactivity, loud/abdominal breathing, and ptyalism) on study day 35 most likely due to reflux at dosing.
Two females were sacrificed for absence of delivery on day 25 p.c.: one female (300 mg/kg/day group) and one female (1000 mg/kg/day group). These females were not pregnant.

CLINICAL SIGNS:
Hypoactivity, loud/abdominal breathing and dyspnea were observed at 1000 mg/kg/day and in males only (except hypoactivity which was also noted in one female treated at 1000 mg/kg/day, during the lactation period). These clinical signs appeared mainly from study day 30 with the exception of one male with loud breathing from study day 9 and abdominal breathing from study days 9 to 12. All these clinical signs were considered to be related to the treatment with the test item and of toxicological significance.
Ptyalism was considered to be related to the test item but of minor toxicological importance.
A treatment-related effect was considered to be unlikely for the few others clinical signs commonly observed in this species and strain.

BODY WEIGHT (GAIN):
There were no biologically significant effects on mean body weight or mean body weight changes during the premating, mating, gestation or lactation periods.
In males, there were a few isolated and statistically significant increases in mean body weight changes. In absence of any dose relationship and taking into account the amplitudes of the changes, a relationship to the treatment with the test item was considered unlikely.

FOOD CONSUMPTION:
There were no effects on mean food consumption during the premating, mating, gestation or lactation periods.

NEUROBEHAVIOURAL EXAMINATION:
Functional Observation Battery
There were no relevant differences in treated groups when compared with control group.

Motor activity
There were no relevant differences in motor activity (horizontal movements and rearing) in treated groups when compared with control group.

HAEMATOLOGY:
When compared with controls, a few statistically significant differences were recorded from animals treated at 300 or 1000 mg/kg/day (increased white blood cells, decreased neutrophils and decreased fibrinogen). These findings were of small amplitude and observed in one sex only or with opposite trends between sexes. Therefore, these findings were considered to be of non toxicological significance.

CLINICAL CHEMISTRY:
Overall, when compared with controls there were a few statistically significant differences which were considered to be of non toxicological significance taking into account the amplitudes of the changes, the absence of a dose-relationship and/or the absence of a similar correlate in the other sex.

URINALYSIS:
There were no effects on mean urinalysis parameters.

REPRODUCTIVE PERFORMANCE:
There were no treatment-related effects on mating and fertility data.
All animals mated within comparable mean number of days taken to mate.
All females were pregnant, except one at 300 mg/kg/day and one at 1000 mg/kg/day.

ORGAN WEIGHTS:
There were no changes in organ weights attributed to the test item administration.
Organ weight changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, and/or were not dose-related in magnitude.

GROSS PATHOLOGY:
Unscheduled sacrifices
High-dose male was sacrificed moribund on study day 35. At necropsy, the lungs were enlarged and showed focal white discoloration (approximately 2.5 cm long, 1.5 cm wide).
High-dose female was not pregnant and was sacrificed on day 25 p.c. At necropsy, there was a transverse vaginal septum and enlarged uterine cervix.
Mid-dose female was not pregnant and was sacrificed on day 25 p.c. The only macroscopic finding was red discoloration of the thymus. This is a common finding noted at necropsy in rats, which correlated with hemorrhages and is considered to be related to the sacrifice procedure.

Terminal sacrifice
The few macroscopic findings noted at the end of the treatment period were of those commonly recorded in the Sprague-Dawley rat and none were considered to be related to the test item administration.
Red discoloration of the thymus was noted in a few test item-treated males and a single mid-dose female. This is a common finding noted at necropsy in rats, which correlated with hemorrhages and is considered to be related to the sacrifice procedure. Therefore it was not considered to be related to the test item administration.
Cutaneous scabs were noted in a few test item-treated males and in a single high-dose female. However since this is a common finding in rats, it was considered to be incidental and unrelated to the test item administration.

HISTOPATHOLOGY:
Unscheduled sacrifices
The cause of death of high-dose male was acute bronchioalveolar inflammation which was associated with fragments of alimentary content and numerous bacterial colonies. This acute inflammation was characterized by edema, correlated with macroscopic enlargement and white discoloration of the lungs, and was associated with hemorrhages. This pulmonary inflammation was most likely secondary to the reflux at dosing noted clinically.
In high-dose female, the transverse vaginal septum was confirmed microscopically since there was a thin band of connective tissue covered by a mucous epithelium continuous with the vaginal epithelium. There was marked mucification of the vaginal epithelium and accumulation of mucus in the vaginal lumen. The transverse vaginal septum is a developmental anomaly occasionally observed in female rats (De Schaepdrijver et al., 1995; Lezmi et al., 2011). The presence of this transverse vaginal septum explained that this female was unable to breed.

In mid-dose female, there were no significant microscopic findings in the reproductive organs or other tissues. This female was at the end of estrus/early metestrus.

Terminal sacrifice
There were no microscopic changes attributed to the test item administration. There were no significant changes in the female and male reproductive organs.
All microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the Sprague-Dawley rat.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

MORTALITY/VIABILITY:
There were no obvious treatment-related effects on pup mortality.

CLINICAL SIGNS:
3.5.2 Clinical signs (Appendix 31)
There were no clinical signs in pups that could be considered to be treatment-related.

The few clinical signs recorded concerned a few pups:
Control group:
- one pup from litter had an hematoma on neck (days 1 to 4 p.p.),
100 mg/kg/day group:
- one pup from litter had an hematoma on back (days 1 to 3 p.p.),
- one pup from litter was emaciated and cold to the touch (day 5 p.p.),
1000 mg/kg/day group:
- one pup from litter had a scab on back (from day 3 p.p.),
- one pup from litter had an hematoma on head (days 1 to 4 p.p.),
- one pup from litter had an hematoma on back (days 2 to 4 p.p.),
- one pup from litter was emaciated and cold to the touch (day 5 p.p.).

BODY WEIGHT (GAIN):
There was a moderate increase in mean pups body weight at 1000 mg/kg/day. However, taking into account the amplitude of this change, it was considered that there were no toxicologically significant effects on mean body weight of pups during the lactation period (day 1 or day 5 p.p.).

Effect levels (F1)

open allclose all
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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

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 NOEL for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day,
- the No Observed Adverse Effect Level (NOAEL) 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 following daily oral administration (gavage) to male and female rats from before mating, during mating and, for the females, throughout gestation until day 5 post‑partum (p.p.) inclusive.

This study provides information on the 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.

Methods

Three groups of ten male and ten female Sprague-Dawley rats received the test item, daily, by oral administration (gavage), before mating, during mating and, for the males, until sacrifice, for the females, throughout gestation until day 5p.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, corn oil, under the same experimental conditions. The dosing volume was 5 mL/kg/day.

 

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 5p.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.

A Functional Observation Battery including touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, rectal temperature and motor activity was performed on five males and females per group at the end of the study. Prior to sacrifice, blood samples were also taken from these animals for analysis of hematology and blood biochemistry parameters.

 

The males were sacrificed after completion of the mating period. Body weights and selected organs weights were recorded and a complete macroscopic post-mortem examination performed, with particular attention paid to the reproductive organs. A microscopic examination was also conducted on selected organs from the first five males in the control group and the high-dose group. Microscopic examination was conducted on all macroscopic lesions from all groups.

 

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.

 

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


Results

The test item concentrations in the administered dosage forms were within an acceptable range of variation (± 15%). The test item was not detected in control samples.

 

Mortality

There were no unscheduled deaths in control, 100 and 300 mg/kg/day groups. At 1000 mg/kg/day, one male was sacrificed moribund (hypoactivity, loud/abdominal breathing, and ptyalism) on study day 35 most likely due to reflux at dosing.

 

Clinical signs

Hypoactivity, loud/abdominal breathing and dyspnea were observed at 1000 mg/kg/day and in males only (except hypoactivity which was also noted in one female treated at 1000 mg/kg/day, during the lactation period). These clinical signs were considered to be related to the treatment with the test item. Ptyalism was considered to be related to the test item but of minor toxicological importance.

 

Body weight and body weight change

There were no biologically significant effects on mean body weight or mean body weight changes during the premating, mating, gestation or lactation periods.

 

Food consumption

There were no effects on mean food consumption during the premating, mating, gestation or lactation periods.

 

Functional Observation

There were no relevant differences in treated groups when compared with control group.

 

Motor activity

There were no relevant differences in motor activity (horizontal movements and rearing) in treated groups when compared with control group.

 

Hematology

There were nofindings of toxicological significance.

 

Blood biochemistry

There were nofindings of toxicological significance.

 

Urinalysis

There were no effects on mean urinalysis parameters.

 

Mating and fertility data

There were no treatment-related effects on mating and fertility data. All animals mated within comparable mean number of days taken to mate.

 

Delivery data

There were no relevant differences between control and test item-treated groups (mean duration of gestation, mean number of corpora lutea, mean number of implantations, mean pre‑implantation loss, mean number of pups delivered and mean post-implantation loss).


Pups mortality

There were no obvious treatment-related effects on pup mortality.

 

Pups clinical signs

There were no clinical signs in pups that could be considered to be treatment‑related.

 

Live birth, viability and lactation indexes

There were no treatment-related effects on live birth, viability and lactation indexes.

 

Pup body weight

There were no toxicologically significant effects on mean body weight of pups during the lactation period (day 1 or day 5 p.p.).

 

Sex ratio

There were no treatment-related effects on sex ratio (% of male pups) both on days 1 and 5 p.p.

 

Pathology:    

- Unscheduled sacrifices: the cause of death of thehigh-dose male sacrificed moribund on study day 35 was acute bronchioalveolar inflammation most likely secondary to the reflux at dosing noted clinically. One high-dose female was not pregnant and was sacrificed on day 25 p.c.. At necropsy, there was a developmental anomaly of the vagina (transverse septum) which explained that this female was unable to breed. One mid-dose female was not pregnant and was sacrificed on day 25 p.c. There were no significant macroscopic and microscopic changes in this female.

- Terminal sacrifice: The test item administration at 100, 300 or 1000 mg/kg/day did not induce any organ weight, macroscopic or microscopic changes.

 

Conclusion

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

 

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 NOEL for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day,

the No Observed Adverse Effect Level (NOAEL) 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.