Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River (Europe) Laboratories Inc, Toxicoop Ltd. Hungary, 1103 Budapest, Cserkesz u. 90
- Age at study initiation: 10-11 weeks old at starting, 12-13 weeks old at mating
- Weight at study initiation: 351-414 g (males), 204-261 g (females)
- Housing: type II and III polypropylene/polycarbonate
- Diet: ssniff SM R/M-Z+H "Autoclavable complete feed for rats and mice - breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany, ad libitum
- Water: tap water from municipal supply, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19.9-24.0 °C
- Humidity: 30-60 %
- Air changes: 15-20 changes/hour
- Photoperiod: 12 hours dark / 12 hours light

IN-LIFE DATES: From: 29 October 2009 (animal arrival) To: 25 December 2009 (last necropsy)
Route of administration:
oral: gavage
Vehicle:
other: 0.1 % Tween 80 in PEG 400 (w/v)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle at the appropriate frequency to allow the use of formulations within 3 days while stored at 5 ± 3 °C.

VEHICLE
- Justification for use and choice of vehicle: the test item is not soluble in water. According to the analytical method, a mixture of 0.1 % Tween 80 in PEG 400 (w/v) was considered a suitable vehicle to be used for the preparation of dose formulations for oral administration.
- Concentration in vehicle: 0, 18.75, 75 and 250 mg/mL
- Amount of vehicle: 4 mL/kg bw
- Lot/batch nos.: 1435799 (PEG 400), 1435771 (Tween 80)
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually. Bedding material suitable for nesting was provided.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Top, middle and bottom samples were taken for analysis of concentration and homogeneity, in duplicate from each of the test item dosing formulations (low, mid and high dose) on 3 occasions, approximately during the first, mid and last week of treatment as practical. Similarly, one sample was taken in duplicate from the control solution for concentration measurements.
Duration of treatment / exposure:
Main males were dosed for at least 28 days (14 days pre-mating and 14 days mating plus an optional extended post-mating period), then they were euthanized and subjected to necropsy examination or alternatively were retained and continued to be dosed for the possible conduction of a second mating if considered appropriate.
Main females were dosed for 14 days pre-mating, for up to 14 days mating, through gestation period and up to and including the day before necropsy (at least 4 days post-partum dosing). Females showing no evidence of copulation were sacrificed 24-26 days after the last day of the mating period.
Recovery animals (not used for the assessment of reproduction/developmental toxicity) were kept at least for further 14 days after the first scheduled euthanasia of dams, without treatment to detect delayed occurrence, persistence of or recovery from toxic effects.
All F1 offspring was terminated on postpartal day 4 or shortly thereafter.
Frequency of treatment:
Once daily
Details on study schedule:
- Age at mating of the mated animals in the study: 12 weeks old
Remarks:
Doses / Concentrations:
0, 75, 300 and 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
Main study: 12 animals
Recovery: 5 animals (control and high dose only; not used for the assessment of reproduction/developmental toxicity)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: doses were selected based on a preceding dose range finding study (LAB Ltd., study code 09/174-220PE).
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: once daily, after treatment

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: once weekly, after treatment

BODY WEIGHT:
- Time schedule for examinations:
Main study: parent animals were weighed on the first day of dosing (Day 0), at least weekly thereafter and at termination. Parent females were weighed on gestation Days 0, 7, 14 and 20 and on postpartal Days 0 and 4. Body weight of females was additionally weighed on gestation Days 10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: at least weekly

OTHER: number of pairings, fertile pairings, infertile males, pregnant females, sperm positive but non-pregnant females, non-mated females, corpora lutea/dam, implantations/dam and dams with live pups on Day 0 and 4, duration of pregnancy, pre-implantation mortality, intrauterine mortality and total intra- and extrauterine mortality.

Various ophthalmology, clinical pathology, urinalysis and neurobehaviour parameters were assessed in parental animals according to OECD guideline 422; please refer to "7.5.1 Repeated dose toxicity: oral".
Oestrous cyclicity (parental animals):
Examination of the oestrous cycle in parental females was performed during the mating period, until a sperm positive vaginal smear or a vaginal plug was identified.
Sperm parameters (parental animals):
Parameters examined in male parental generation: testis weight, epididymis weight, weight of seminal vesicles with coagulating glands as well as histological examination of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: mean pup body weight on postnatal Days 0 and 4, mean pup body weight gain (per litter) between postnatal Days 0 and 4, number of live births per litter and number of viable pups per litter on postnatal Days 0 and 4

GROSS EXAMINATION OF DEAD PUPS:
yes, at least for external abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals, after at least 28 days of dosing
- Maternal animals: all surviving animals, after at least 4 days of postpartum dosing

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Samples for histopathology were collected from 5 parental animals/sex/group. The following organs and tissues (or representative samples) were preserved: adrenals, aorta, brain, epididymides, eyes with optic nerve, oesophagus, femur with marrow, heart, kidneys, large intestine, lachrymal gland with Harderian glands, liver, lungs with bronchi, lymph nodes, mammary gland, ovaries with oviduct, pancreas, pituitary, prostate, salivary gland, sciatic nerve, seminal vesicles with coagulating glands, skeletal muscle, skin and subcutis, small intestine, spinal cord, spleen, sternum with marrow, testes, thymus, thyroid with parathyroids, tongue, trachea, urinary bladder, uterus and vagina.
Histological examinations were performed in control and high dose animals only. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

At the time of termination, body weight and weight of the following organs of all parental animals were determined:
uterus (with and without cervix), vagina, testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands, brain, ovaries and pituitary gland
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed on postnatal Day 4.
- These animals were subjected to postmortem macroscopic examination.

GROSS NECROPSY
- Pups euthanised at postnatal Day 4 were carefully examined at least externally for gross abnormalities. Any found dead pups were subjected to necropsy with internal and external macroscopic examinations.
Statistics:
The statistical evaluation was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons was performed using Mann-Whitney U-test. Chi square test was performed where applicable.
Reproductive indices:
Male and female mating indices, male and female fertility indices and gestation index
Offspring viability indices:
Survival index on postnatal Days 0 and 4, sex ratio on postnatal Days 0 and 4
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:
effects observed, treatment-related
Description (incidence and severity):
transient liver and kidney weight changes
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY
There was no mortality or relevant clinical findings.

BODY WEIGHT AND FOOD CONSUMPTION
Body weight and food consumption were not affected by the treatment with the test item.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no test item related differences in the oestrous cycle evaluated during the mating period.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There was no test item-related increase in infertile males.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No effects were noted on mating, fertility and gestation indices.

ORGAN WEIGHTS
In main male animals, statistically higher mean liver weights were noted at 300 and 1000 mg/kg bw/d, with an apparent dose response, and higher kidney weights were noted at 1000 mg/kg bw/d, as absolute values (p<0.01) and when adjusted for body and brain weight relative values which showed statistical significance at 75 mg/kg bw/d (p<0.05). Similar effects were observed in females (p<0.05 for absolute kidneys weight at 1000 mg/kg bw/d; p<0.01 for absolute liver weight at 300 and 1000 mg/kg bw/d) and the brain and body weight relative liver weights were also statistically higher than control at 300 and 1000 mg/kg bw/day. No statistically significant variations were noted in the absolute or relative liver or kidneys weights after a 14-day recovery period in the female animals; in the recovery male animals, the relative mean liver weight referring to body weight was slightly higher than control at 1000 mg/kg bw/d.
Although these organ weight changes were considered potentially related to test item administration, in the absence of any concomitant clinical, clinical pathological, macroscopical, histopathological or physiological reproductive adverse effects, these generally transient liver and kidney weight changes were regarded as adaptive rather than reflecting a toxic response.

GROSS PATHOLOGY
There were no test item-related gross lesions.

HISTOPATHOLOGY
There was no microscopical evidence of test item-related findings.

OTHER FINDINGS
No test item-related changes were noted in clinical laboratory, urinalysis, ophthalmoglogical, neurobehavioural and developmental parameters.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no reproductive effects were noted
Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
There were no statistically significant variations noted in the pre/postimplantation loss and total intrauterine mortality values in the treated animals compared to control animals.

BODY WEIGHT (OFFSPRING)
Body weights at birth and at post natal Day 4 was not affected by the treatment with the test item.

SEXUAL MATURATION (OFFSPRING)
There were no changes in the sex ratio.

GROSS PATHOLOGY (OFFSPRING)
No macroscopic changes were seen in F1 offspring generation euthanized and examined at scheduled termination.
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no F1 offspring effects were noted
Reproductive effects observed:
not specified

Dose Formulation Analysis:

Dose formulations were homogenous. The measured concentrations varied from 99 to 103 % of nominal concentrations and these results were considered suitable for the study purposes. Assessment of test item stability in the vehicle, 0.1% Tween 80 in PEG 400 (w/v), (LAB study code 09/174-316AN) indicated an up to 24-hour stability at room temperature, and 72 -hour stability at 5 ± 3 °C, at concentrations from approximately 1 to 250 mg/mL in the vehicle with a recovery within the acceptable range of 100 ± 10 %.

Executive summary:

In a subacute reproduction / developmental toxicity screening study (Kubaszky, 2010a) Montanol 800 (≥98 %) in 0.1 % Tween 80 in PEG 400 (w/v) was administered to 12 Wistar rats/sex/dose level by oral gavage (4 mL/kg bw) at dose levels of 0 (vehicle only), 75, 300 or 1000 mg/kg bw/d. Unmated recovery animals (5 rats/sex at control and high dose) were kept for further 14 days, after the first scheduled necropsy of dams, without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects. In the reproductive/developmental toxicity screening part of the study, possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and on the development of the F1 offspring from conception to day 4 post partum were assessed. There were no test item-related effects in mortality, clinical signs, body weight, food consumption, ophthalmology, haematology, clinical chemistry, urinalysis, neurobehaviour, gross pathology, histopathology, reproductive or developmental endpoints. Transient liver and kidney weight changes noted at 300 and 1000 mg/kg bw/d were considered to represent adaptive responses. Accordingly, the NOEL for effects on reproduction in males and females was 1000 mg/kg bw/d.

This screening study is acceptable and satisfies the requirement for test guideline OECD 422.

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
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a subacute reproduction / developmental toxicity screening study the registration substance (≥98 %) in 0.1 % Tween 80 in PEG 400 (w/v) was administered to 12 Wistar rats/sex/dose level by oral gavage (4 mL/kg bw) at dose levels of 0 (vehicle only), 75, 300 or 1000 mg/kg bw/day. Unmated recovery animals (5 rats/sex at control and high dose) were kept for further 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects. In the reproductive/developmental toxicity screening part of the study, possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and on the development of the F1 offspring from conception to day 4 post partum were assessed. There were no test item-related effects in mortality, clinical signs, body weight, food consumption, ophthalmology, haematology, clinical chemistry, urinalysis, neurobehaviour, gross pathology, histopathology, reproductive or developmental endpoints. Transient liver and kidney weight changes noted at 300 and 1000 mg/kg bw/d were considered to represent adaptive responses. Accordingly, the NOEL for effects on reproduction in males and females was 1000 mg/kg bw/d.

There is no two-generation reproduction toxicity study available. However, as there was no indication of impaired fertility up to the limit dose of 1000 mg/kg bw/d in the OECD TG 422 study, no indication of developmental toxicity in the OECD TG 414 prenatal developmental toxicity study, and also no indications of effects on the reproductive system or histopathological changes on reproductive organs including sperm motility in the 90 -day subchronic oral toxicity study, no concern with regard to the endpoint `fertility` is derived. Based hereupon and taking the exposure profile into account, additional testing for this endpoint is waived (see detailed justification in section 7.8.1.)


Short description of key information:
In a subacute reproduction / developmental toxicity screening study in male and female Wistar rats, there were no effects on fertility following daily oral gavage at up to and including 1000 mg/kg bw/day. The NOAEL for fertility (oral exposure) was considered to be greater than 1000 mg/kg bw/day. No indications of effects on the reproductive system or histopathological changes of reproductive organs including sperm motility were derived in a 90-day subchronic oral toxicity study.

Justification for selection of Effect on fertility via oral route:
Guideline study according to GLP

Effects on developmental toxicity

Description of key information
A guideline conform prenatal developmental toxicity study according to OECD TG 414, repeated administration of the registration substance via oral gavage from gestation day 5 to 19 to rats, revealed no indications of embryo/foetotoxicity or teratogenicity. The NOAEL was 1000 mg/kg body weight per day. Additionally, no effects on reproductive parameters were revealed in a reproduction / developmental toxicity screening study in male and female Wistar rats in an OECD TG 422 study following daily oral gavage at up to and including 1000 mg/kg bw/d. Furthermore, no effects on the reproductive system or histopathological changes in reproductive organs including sperm motility were observed in a 90-day subchronic oral toxicity study according to OECD 408.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-04-11 to 2014-07-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study according to GLP
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und dLebensmittelsicherheit, München, Germany)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Test System
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: approx. 12-13 weeks old
Body weight at the allocation of the animals to the experimental groups:interval within ± 20% of the mean weight, if technically possible.
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act
on Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3°C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0902)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological
controls at regular intervals)
- The animals were kept individually in IVC cages (except during the mating period when two females were paired with one male), type III H,
polysulphone cages on Altromin saw fibre bedding (lot no. 230113)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days)

Number and Sex of the Animals
156 animals (52 males and 104 females) were included in the study.

Preparation of the Animals
After the acclimatisation period of at least 5 days, females were paired with males as per the ratio of 1:2 (male to female). Prior to the start of the
mating a detailed clinical observation outside the home cage was made. None of the animal showed pathological signs before the initiation of
mating period.
Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Details on exposure:
The test item was weighed into a tarred plastic vial on a precision balance and the required volume of sesame oil was added and further
vortexing it for 2-3 minutes.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration.
The test item formulation was prepared freshly on each administration day before the administration procedure.
The vehicle was also used as control item.

The following doses were evaluated:
Control: 0 mg/kg body weight
Low Dose: 100 mg/kg body weight
Medium Dose: 300 mg/kg body weight
High Dose: 1000 mg/kg body weight

The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence
of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL. The doses were selected on the basis of data
from a Dose Range Finding Study (BSL study no. 130404).

The animals in the control group were handled in an identical manner to the test group subjects and received sesame oil using the same
volume as used for the high dose group.

The test item formulation or vehicle was administered at a single dose to the animals by oral gavage.
The application volume for all groups was 4 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The assessment of homogeneity as well as a determination of the nominal concentration of the test item in the vehicle were performed at
various intervals.
Samples for analysis of the dose formulations of the test item in the vehicle (nominal concentration) were taken in the first and last week of the
study for all doses.
Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation. Samples were taken in the first and last week of the study.
All formulation samples were stored at -20° C and were analysed after completion of the in-life phase of the toxicity study at BSL BIOSERVICE
Scientific Laboratories GmbH.
Details on mating procedure:
Females were paired for cohabitation in batches in order to regularise the number of animals for terminal sacrifice on a particular day.
The subsequent morning and the next morning onwards, the vaginal smear of female was checked to confirm the pregnancy.
The day on which sperms were observed in the vaginal smear was considered as gestation day ‘0’. Mated females were assigned in an unbiased
manner to the control and treatment groups ensuring that group mean body weights are comparable with each other.
Each animal was assigned a unique identification number. 52 males and 104 females were included in the study instead. Out of 104 females,
95 were mated and distributed 24 each in control, MD, HD and 23 in LD group. Non mated females were discarded without any further observations.
Duration of treatment / exposure:
The test item was orally administered daily in graduated doses to several groups of pregnant females from the gestation day (GD) 5
to gestation day (GD) 19
Frequency of treatment:
Once per day. 7 days per week
Duration of test:
On GD 20, i.e. the day prior to the expected day of delivery, the presumed pregnant females are subjected to a caesarean section.
No. of animals per sex per dose:
156 animals (52 males and 104 females) were included in the study. The 4 groups comprised 24 each in control, MD, HD and 23 in LD group. Five non mated females were discarded without any further observations.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL. Selection was based on a preliminary dose-range-finding study.
- Rationale for animal assignment (if not random): Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that the mean body weights were comparable to each other
Maternal examinations:
Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly
during the treatment.
The sperm positive females were weighed during gestations days 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except
on the day of their arrival.
Food consumption of pregnant females was measured on gestations days 5, 8, 11, 14, 17 and 20.
Food consumption was measured neither for males during the entire study nor for both male and females during the mating period.

Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of
anticipated effects after dosing. The health condition of the animals was recorded. At least once daily all animals were observed for morbidity
and mortality.
Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Post-Mortem Examination
On gestation day 20, sperm positive were subjected to a caesarean section after sacrificing the animals using an overdose of pentobarbital
injected intraperitoneally (Narcoren®, Merial; lot no.: 228112; expiry date: 30/11/2015).
At the time of termination, the dam (presumably pregnant female) was examined macroscopically for any structural abnormalities or
pathological changes which may have influenced the pregnancy.
Immediately after the termination or as soon as possible after death, the uteri were removed and the pregnancy status of the dams was confirmed.
Uteri that appear non-gravid were further examined by staining with 10 % ammonium sulphide solution to confirm the non-pregnant status.
Each gravid uterus with the cervix was weighed.
The number of corpora lutea was counted for pregnant animals. The uterine contents were examined for embryonic or foetal deaths as well as
the number of viable foetuses. The degree of resorption (late and early) was confirmed in order to help estimate the relative time of death
of the conceptus. The position and number of foetuses in each uterine horn was also recorded.
Males were sacrificed without any observations any time after the mating.

Foetal Evaluations
All foetuses from a particular dam were identified by using different colour strings and were weighed and sexed based on the anogenital distance.
Each foetus was examined for external anomalies.
One half of each litter was processed by Alizarin red staining and examined for skeletal alterations. The remaining litter was examined for soft tissue anomalies by a microdissection technique.
Craniofacial examination of the heads of the foetuses used for the soft tissue examination was performed for internal structure including the eyes,
brain, nasal passage and tongue by razor blade serial sectioning technique.
For interpretation of significant external, visceral, and skeletal findings, they were described as developmental variations (alterations in anatomic
structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence,
representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or
interfere with normal body function, or may be incompatible with life).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
A statistical assessment of the results of the body weight, food consumption was performed by comparing values of dosed with control animals
using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters
were analysed using a Chi-square test. The statistics are performed with GraphPad Prism V.6.01 software or IDBS Workbook 8.1.2, ANOVA v8.8
software (p<0.05 is considered as statistically significant).
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
All sperm positive females survived until the scheduled necropsy.
Minor clinical signs like moving the bedding , piloerection , salivation were observed on few isolated days thoughout the dose groups. Additionally, few spontaneous clinical signs, including alopecia , eschar and slight nasal discharge were observed in all dose groups including control animals. These various clinical signs in the control and treatment groups were not considered to be adverse.
None of the female showed signs of abortion or premature delivery prior to the scheduled sacrifice.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Prenatal Data
The statistical analysis of prenatal data revealed no significant effect on prenatal parameters like gravid uterus weight, number of corpora lutea,
live foetuses, dead foetuses, resorptions (early and late), sex ratio, percent preimplantation loss, post implantation loss, group mean number of male and female foetuses in the treated groups when compared with controls. However, a statistically significant decrease in group mean terminal
body weight and adjusted maternal weight was observed in HD group when compared to the control. This effect on terminal body weight and adjusted maternal weight in HD group could be attributed to treatment with test item.

Litter Data
No statistically significant and treatment-related effects were observed on group mean litter weight, total litter weight, male litter weight,
female litter weight, group mean number of live foetuses, number of males and number of females were observed when compared to the
control group.

Foetal Evaluation
No gross external abnormalities were seen in control and treatment groups.
Skeletal examination of the Alizarin red stained foetuses revealed a range of findings which were of a type or which occurred at an incidence
comparable/ lower in treated groups when compared to the control group. As these skeletal findings were minor variations and due to the lack of dose dependency and consistency in these findings indicates no treatment-related adverse effects. There was no indication of a test item-related trend in the type and incidences of other findings and they were therefore, considered to be spontaneous in nature.
Internal examinations of the foetal viscera by the free-hand-microdissection technique revealed a range of visceral abnormalities in all groups
including the control. All observed findings are considered minor variations, and due to lack of dose dependency no significant toxicological significance can b attributed to these findings and they are considered to be spontaneous in nature.
Craniofacial examination by a razor blade serial sectioning technique revealed a range of visceral findings in all groups including controls. These findings are not considered as treatment-related and solely spontaneous in nature.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
In this prenatal developmental toxicity study, the repeated dose administration of 1-Hexanol, 2 ethyl-, manuf. of, by products from, distn Residues
to pregnant female Wistar rats at doses of 100, 300 and 1000 mg/kg body weight from gestation day 5 to 19 revealed no major toxicological findings in terms of mortality but few clinical signs in MD and HD group and statistically significant effects on female body weight and food consumption was observed in HD group.
In foetuses, no test item related effect on foetal parameters was observed in treatment groups when compared with the controls.
There were few skeletal findings mainly related to ossification observed and evenly distributed between all dose groups including controls.
These skeletal findings are considered to be minor variations which are not associated with long term consequences on survival, general growth and development. Due to the lack of dose dependency and consistency, these skeletal findings were not considered to be treatment-related.

Based on the findings from this study, The NOAEL of 1-Hexanol, 2 ethyl-, manuf. of, by products from, distn Residues in Wistar rat for
maternal toxicity is considered to be 300 mg/kg body weight/day and for foetal toxicity as 1000 mg/kg body weight/day.
Executive summary:

For the registration substance a guideline conform OECD 414 prenatal developmental toxicity study was performed following the principles of GLP. The aim of this study was to assess possible adverse effects on pregnant females and embryo-foetal development which could arise from repeated exposure of 1-Hexanol, 2 ethyl-, manuf. of, by products from, distn Residues, via oral administration (gavage) to female rats during the gestation days 5 to 19. Nulliparous and non-pregnant females were mated with males (2:1 ratio) and divided into four groups based on their body weights on the day of sperm positive vaginal smears (GD 0). The 4 groups comprised 24 animals each in control, MD, HD and 23 in LD group. Animals of the control group were handled identically as the dose groups, but received sesame oil, the vehicle used in this study.

Analysis of formulation samples for nominal concentration and homogeneity revealed stable and homogenious formulations over the period they were used

During the period of administration and also during pre-exposure gestation days (0-4), the animals were observed precisely each day for signs of toxicity and mortality. All female animals were sacrificed on the respective gestation day 20. Following the gross necropsy, the uteri and ovaries were removed, weighed and examined for number of implantations, resorptions (early and late) live and dead foetuses. Foetuses were identified by color strings, sexed and weighed. All foetuses were observed for external abnormalities, half of the Foetuses for visceral and craniofacial abnormalities and the remaining half of the litter was observed for skeletal abnormalities. The sperm positive females were weighed during gestations days 0, 5, 8, 11, 14, 17 and 20. Food consumption of sperm positive females was measured on gestations days 5, 8, 11, 14, 17 and 20. The uteri of the non-pregnant females were processed with 10 % ammonium sulphide solution and checked for the early embryonic deaths.

The following doses were evaluated:

Control:                        0         mg/kg bw/day

Low Dose:                    100     mg/kg bw/day

Medium Dose:              300    mg/kgbw/day

High Dose:                   1000  mg/kgbw/day

The test item formulation was prepared freshly on each day of administration. The test item was suspended in sesame oil and administered daily during the gestation days 5 and 19 to female animals.

Summary Results

All sperm positive females survived until the scheduled necropsy.

Minor test item-related clinical signs like moving the bedding, piloerection and salivation were observed on few , isolated days in all dose groups. There were also few sponstaneous clinical signs like alopecia, eschar and slight nasal discharge observed. All of these findings are considered to be not adverse. None of the female showed signs of abortion or premature delivery prior to the scheduled sacrifice.

Statistical analysis of body weight and body weight gain data during gestation period revealed statistically significant decrease in body weight on gestation day 11, 17 and 20 in HD group when compared with the controls. Overall body weight gain during 0-20 in HD group was also decreased (although statistical significance was not achieved) when compared with controls. In correlation to the body weight and body weight gain, the food consumption was comparable in the C, LD and MD groups. However, in HD group, statistically significant decrease in food consumption was observed from GD 5 throughout the study period when compared with controls.

The statistical analysis of prenatal data revealed no significant effect on prenatal parameters like gravid uterus weight, number of corpora lutea, live foetuses, dead foetuses, resorptions (early and late), sex ratio, percent preimplantation loss, post implantation loss, group mean number of male and female foetuses in the treated groups when compared with controls.

No statistically significant and treatment-related effects were observed on group mean litter weight, total litter weight, male litter weight, female litter weight, group mean number of live foetuses, number of males and number of females were observed when compared to the control group.

No gross external abnormalities were seen in control and treatment groups.

Skeletal examination of the Alizarin red stained foetuses revealed a range of findings which were of a type or which occurred at an incidence comparable/ lower in treated groups when compared to the control group. However, all of these skeletal findings were minor variations which are not associated with long term consequences on survival, general growth and developmentand. Due to the lack of any dose dependency, these skeletal findings were not considered to be treatment-related. Internal examinations of the foetal viscera by the free-hand-microdissection technique revealed a range of visceral abnormalities in all groups including the control. .


Conclusion

In this prenatal developmentaltoxicity study,the repeated dose administration of 1-Hexanol, 2 ethyl-, manuf. of, by products from, distn Residues to pregnant female Wistar rats at doses of 100, 300 and 1000 mg/kg body weight from gestation day 5 to 19 revealed no major toxicological findings in terms of mortality but few minor clinical signs in MD and HD group not considered to be adverse. Statistically significant effects on female body weight and food consumption were observed in animals from the HD group. 

In foetuses, no test item related effect on foetal parameters was observed in treatment groups when compared with the controls. There were few skeletal findings mainly related to ossification observed and evenly distributed between all dose groups including controls. These skeletal findings are considered to be minor variations which are not associated with long term consequences onsurvival, general growth and development. Due to the lack of dose dependency and consistency, these skeletal findings were not considered to be treatment-related.

Based on the findings from this study, the NOAEL of 1-Hexanol, 2 ethyl-, manuf. of, by products from, distn Residues in Wistar rat for maternal toxicity is considered to be 300 mg/kg body weight/day and for foetal toxicity as 1000 mg/kg body weight/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Data base is considered to be sufficient to evaluate the endpoint of developmental toxicity. No further testing in a second species is considered necessary taking the available data from an OECD TG 414, an OECD TG 421 as well as an OECD TG 408 study into account. Based on the results from these studies, repetitive testing of this endpoint is considered to be not justified for scientific as well as animal welfare reasons.
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 registration substance was investigated for reproductiove toxicity in a guideline conform prenatal developmental toxicity study according to OECD TG 414. Groups of rats were adminstered via oral gavage doses of 0 (vehicle control), 100, 300 or 1000 mg/kg body weight per day from gestation day 5 to 19. No mortality occurred throughout the study period and all sperm positive females survived until scheduled necroscopy. Body weight and body weight gain as well as food consumption was reduced in the high dose female animals. Prenatal data like gravid uterus weight, number of corpora lutea, live or dead foetuses, early and late resorptions, sex ratio, percent preimplantation loss, post implantation loss, group mean number of male and female foetuses, revealed no differences between treated groups and control. Addittionally, no significant differences were observed on group mean litter weight, total litter weight, male litter weight, female litter weight, group mean number of live foetuses in treated groups when compared to the control group. No gross external abnormalities were seen in control and treated groups. Visceral and/or skeletal malformations were not detected in any of the dose groups including control. Based on the findings from this study, the NOAEL of the registration substance for foetal toxicity in rats was considered to be 1000 mg/kg body weight per day.

The findings from the prenatal developmental toxicity study is supported by results from a subacute reproduction / developmental toxicity screening study according to OECD TG 422. Administration to 12 Wistar rats/sex/dose level by oral gavage (up to 54 days of exposure) at dose levels of 0 (vehicle only), 75, 300 or 1000 mg/kg bw/day revealed no effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and on the development of the F1 offspring from conception to day 4 post partum. There were no test item-related effects in mortality, clinical signs, body weight, food consumption, ophthalmology, haematology, clinical chemistry, urinalysis, neurobehaviour, gross pathology, histopathology, reproductive or developmental endpoints. Transient liver and kidney weight changes noted at 300 and 1000 mg/kg bw/d were considered to represent adaptive responses. Accordingly, the NOEL for developmental and/or teratogenic effects was considered to be 1000 mg/kg bw/day.

No effects indicative of reproductive toxicity were also revealed in a 90 -day subchronic oral toxicity study in rats according to OECD TG 408. Neither effects relevant for the reproductive system nor histopathological changes in reproductive organs including sperm motility could be observed up to the highest dose of 750 mg/kg body weight per day tested.

No prenatal developmental toxicity study on a second species (e.g. rabbits) is available. However, because no indications of embryo-/foetotoxic effects and/or teratogenic properties have been revealed in separate guideline conform studies up to the limit dose of 1000 mg/kg body weight per day as well as no histopathological changes of reproductive organs or effects on sperm motility were observed in a 90 -day subchronic oral toxicity study, repetitive testing of this endpoint in a second species is not considered justified on scientific as well as animal welfare reasons.


Justification for selection of Effect on developmental toxicity: via oral route:
Guideline study according to GLP.

Justification for classification or non-classification

The registration substance is not subject to classification and labelling requirements according to Directive 67/548/EEC (DSD) and Regulation 1272/2008/EC (CLP) with respect to reproductive toxicity. All availbale test results did not indicate any embryo-/foetotoxic and/or teratogenic potential up to the limit dose of 1000 mg/kg body weight per day. Additionally, no indications of a general reproductive toxic potential including fertility were revealed. From a 90 -day subchronic oral toxicity study there are also no histopathological changes in reproductive organs including sperm motility indicative or a reproductive toxic potential.