Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Effect on fertility via the oral route (OECD 422):A 28 day repeat dose oral toxicity study combined with a reproductive/ developmental toxicity screening test was performed in the rat in accordance with GLP and OECD Guideline 422 (Harlan Laboratories Ltd, 2013). The test item was administered by gavage to 3 groups, each of 11 male and 11 female RCCHan™:WIST strain rats for up to 8 weeks (including a 2 week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. There were no treatment related effects observed on mating, fertility, corpora lutea count or gestation length at any dose level. The offspring litter size, sex ratio, viability, growth and development were al comparable to controls and no adverse effects were noted. Since no treatment-related effects were observed for reproduction, a NOAEL was considered to be 1000 mg/kg bw/day. Furthermore, the study showed that the administration of the test item over a period of 28 days did not results in any toxicologically significant effects and hence the NOAEL for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 May 2012 to 15 Feb 2013
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)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: RCCHan™:WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands
- Age at study initiation: 10 weeks
- Weight at study initiation: males: 293 to 342 g, females: 204- 227 g
- Housing: Individually in Markrolon type-3 cages with wire mesh tops and sterilised standard softwood bedding with paper enrichment. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3. Values outside of this range occasionally occured, usually following room cleaning, which was considered not to have any influence on the study.
- Humidity (%): 30-70. Values outside of this range occasionally occured, usually following room cleaning, which was considered not to have any influence on the study.
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 June 2012 To: 20 July 2012
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG300
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared weekly. The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle was added (w/v). The mixtures were stirred using a magnetic stirrer and stored at room temperature (15- 25 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL/day. Dose volume: 5mL/kg bodyweight.
- Lot/batch no. (if required): BCBF5743V
Details on mating procedure:
Mating, gestation and lactation
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if:
-      The daily vaginal smear was positive, or
-      A copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

If a female did not mate during the 14-d period, a second pairing of this female with a male in the same group, which had already mated successfully, was considered. If mating was not recorded during the additional pairing period of a maximum of 14 d, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for infertility.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analysed using a GC-FID method.
Duration of treatment / exposure:
Males: up to 41 d
Females: up to 53 d
Frequency of treatment:
Once daily
Details on study schedule:
Study Sequence
Acclimatisation: 7 d for males and females
First test item administration: Day 1 of pre-pairing for males and females
Pre-pairing: 14 d for males and females
Blood sampling: end of pre-pairing for males and females
Pairing: 14 d maximum for males and females
Gestation: approximately 21 d for females only
Treatment ends: on day of dacrifice for males, on day 3 post partum for females
Necropsy: after treatement of at least 28 d, when no longer needed for assessment of reproductive effects for males. On day 4 post partum (pups on day 4 post partum) for females.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1 (control)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
11/ sex/ dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected based on a previous non-GLP dose range-finsing study in Wistar rats
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatisation and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioural abnormalities in nesting and nursing.

Once prior to the first administration of the test item and weekly thereafter, detailed clinical observations were performed outside the home cage. In females, it was performed on days 0, 6, 13 and 20 of the gestation period. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behaviour were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION: Yes. No food consumption was recorded during the pairing period.
-Males: weekly during pre-pairing and after pairing periods
- Females: pre-pairing period days 1-8 and 8-14; gestation days 0-7, 7-14 and 14-21 post coitum, and days 1-4 post partum.

HAEMATOLOGY: Yes
Blood samples were obtained at the end of the pre-pairing period from 5 males and 5 females from each group. Blood samples were drawn retro-orbitally from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 h before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

The following haematology parameters were determined:

Complete blood cell count
Erythrocyte count, haemoglobin, haematocrit, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, leukocyte count (total), mean corpuscular volume, red cell volume distribution width, mean corpouscular haemoglobin, differential leukocyte count, platelet count.

Coagulation
Prothrombin time (= Thromboplastin time), activated partial Thromboplastin time

Clinical biochemistry
The following clinical biochemistry parameters were determined:
Glucose, urea, creatinine, bilirubin (total), cholesterol (total), triglycerides, aspartate aminotransferase, alanine aminotransferase, alkaline phosphate, gamma-glutamyl-transferase, bile acids, sodium, potassium, chloride, calcium, phosphorous, protein (total), albumin, globulin, albumin/ globulin ratio

OTHER:
FUNCTIONAL OBSERVATION BATTERY (FOB)
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum), relevant parameters from modified Irwin screen test was performed with 5 P generation males and 5 P generation females from each group in place of the usual weekly behavioural observation. This FOB assessment was conducted following the daily dose administration.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151. Activity of the animals (based on beam count) was recorded for 10 minute intervals over a period of 60 min. These data and the total activity over 60 min were reported.
Litter observations:
STANDARDISATION OF LITTERS
The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (wihtout identification) on days 0 (if possible), 1 and 4 post partum.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All survivng animals were sacrificed after treatment of at least 28 d, when no longer needed for the assessment of reproductive effects
- Maternal animals: All surviving animals were sacrificed on day 4 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

GROSS NECROPSY
All animals sacrificed or found dead were weighted and subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4 % formaldehyde solution.

At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital or were sacrificed by CO2 asphyxiation. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

For the parent animals, special attention was directed at the organs of the reproductive system.
The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulphide to visualise possible haemorrhagic areas of implantation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the scheduled sacrifice, the testes and epididymides of all parental males were weighted separately.

In addition, from 5 males and 5 females of each group, sacrificed at the end of the study, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken: adrenal glands (weighed as pairs), brain, heart, kidneys (weighed as pairs), liver, thymus, spleen.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring: All surviving animals were sacrificed on day 4 post partum.

GROSS NECROPSY
Dead pups, except those excessively cannibalised, were examined macroscopically.

All pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
1. Means and standard deviations of various data were calculated.
2. The Dunnett-test (many to one t-test) based on the pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
3. The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
4. Fisher’s exact-test was applied if the variables could be dichotomised without loss of information.
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY
Viability/ mortaility
One male treated with 1000 mg/kg/day was found dead spontaneously on day 12 of the pre-pairing period.

One female treated with 1000 mg/kg/day of the test item was found spontaneously dead on day 11 of the pre-pairing and another female of this group was found dead on day 22 of the gestation period. Two further females of this group were found dead on day 2 of their lactation period.

All these deaths deemed to be caused by aspiration during gavage procedures of massive amount test item and it could be considered as the cause of death, not related to systemic toxicity of the test item. All other animals of this group survived until the scheduled necropsy.

All animals treated with 100 mg/kg/day or 300 mg/kg/day of the test item survived until the scheduled necropsy.

Daily clinical signs or observations
A kinked tail apex with scabs was seen in 1 control male from the treatment day 6 onwards, this was black coloured, additionally, from day 9 onwards. During the pairing period moderate necrosis of this apex was seen additionally. One male treated with 100 mg/kg/day of the test item had slight hair loss on the right shoulder from day 8 and another male of this group had slight breathing noises on one day. One male treated with 1000 mg/kg/day had slight breathing noises on up to 9 days of the pre-pairing period and also on 3 days on the pairing period.

Slight breathing noises and slight hair loss on the head were noted in 1 female treated with 300 mg/kg/day of the test item on single days of the pre-pairing period. Slight breathing noises were noted also in a few females treated with 1000 mg/kg/day on some single days of the pre-pairing and pairing period and slight hair loss on the neck in 1 female of this group. Slightly to moderately ruffled fur was seen in a few females of this group on 3 days. One female neglected its litter and no milk was found in the stomach of the pups during the first litter check. The reason for this is unclear and it was considered to be an incidental finding.

One female treated with 1000 mg/kg/day had a mass on the neck in the gestation period. The mass was still seen during the lactation period. This finding was considered to be incidental.

All these possible findings were considered to be incidental and the slight breathing noises could be possibly treatment-related but not test item-related.

Findings at detailed weekly clinical observations
No toxicological relevant test item-related clinical signs were noted during the weekly observations.

One female treated with 1000 mg/kg/day showed ruffled fur on day 13 and another female of this dose group had a mass on the neck on day 20 post coitum.

FOOD CONSUMPTION, BODY WEIGHT AND WEIGHT GAIN
PRE-PAIRING PERIOD
There were no differences of toxicological relevance between the mean food consumption of test item-related or control males. The mean relative food consumption of test item treated groups was similar to this of the controls.

PRE-PAIRING, GESTATION AND LACTATION PERIODS
Females treated with 1000 mg/kg/day showed a tendency of a slightly decreased mean absolute and relative food consumption. This slight decrease was considered to be not adverse because it is a slight effect without statistical significance and no dose response relationship could be observed.

PRE-PAIRING AND PAIRING PERIODS
There were no differences of toxicological relevance between the mean body weights of test item-treated or control males. The mean body weight gain of all groups compared favourably.

PRE-PAIRING, PAIRING, GESTATION AND LACTATION PERIODS
On some days of the gestation and in the lactation period, mean body weight was slightly decreased (p <0.05) in the 1000 mg/kg/day treatment group when compared to the control animals. This was considered to be not adverse because it was seen on some single days only. No test item-related effects on body weights and body weight gain of females were observed during the pre-pairing and pairing period.

No test item-related effect on body weights and body weight gain of females treated with 100 mg/kg/day or 300 mg/kg/day of the test item were observed during the study.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Eleven, 10, 11 and 10 females in the treatment groups 0, 100, 300 and 1000 mg/kg/day respectively, were mated within the first or second pairing.

The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 3.9, 3.7, 2.4 and 3.2 days in treatment groups 0, 100, 300 and 1000 mg/kg/day respectively. The median precoital time was 2, 3, 2 and 3 days in ascending dose level. For the one control female that mated during the second pairing period, precoital time was 1 d.

The fertility index was 100.0 %, 90.9 %, 100.0 %, 100.0 % and the conception rate showed exactly the same values in the groups treated with 0 mg/kg/day, 100 mg/kg/day, 300 mg/kg/day or 1000 mg/kg/day, respectively.

ORGAN WEIGHTS
No test –item-related changes in the mean organ weights, organ to body weight ratios and organ to brain weight ratios were noted in males and females when compared with the control animals.

GROSS PATHOLOGY
There were no gross lesions that could be attributed to treatment with the test item in sacrificed animals. Although the liver enlargement was recorded in a few males including control group animals, there was no histological correlate. All other gross lesions recorded were considered to be within the range of normal background alterations.

HISTOPATHOLOGY: NON-NEOPLASTIC
The respiratory disorder consisted of necrosis and inflammatory cell infiltration of the trachea, congestion, alveolar edema and alveolar macrophaes of the lung were recorded in dead animals. Major microscopic findings with macroscopic findings in each animal were described as follows:
FINDINGS IN DEAD ANIMALS
Animal No. 34
Trachea: moderate mucosal necrosis and slight inflammatory cell infiltration were recorded and acute reactive and inflammatory changes were considered.
Lung: reactive change consisting of slight congestion and slight alveolar macrophages was recorded. (Macroscopic findings: discolouration, dark red)
Thymus: multifocal slight haemorrhage as lesion during agonal period. (Macroscopic findings: focus/ foci, dark red)

Animal No. 83
Trachea: moderate mucosal necrosis and moderate inflammatory cell infiltration were recorded and acute reactive and inflammatory changes were considered.
Lung: reactive change consisting of moderate congestion, slight alveolar edema and slight alveolar macrophages was recorded. (Macroscopic findings: discolouration, dark red).

Animal No. 87
Trachea: ,oderate mucosal necrosis and moderate inflammatory cell infiltration were recorded and acute reactive and inflammatory changes were considered.
Lung: reactive change consisting of minimal congestion and slight alveolar macrophages was recorded. (Macroscopic findings: discolouration, dark red)
Thymus: multi focal slight haemorrhage as lesion during agonal period. (Macroscopic findings: gelatinous, focus/ foci, dark red)

Animal No.88
Trachea: moderate inflammatory cell infiltration was recorded and acute reactive and inflammatory changes were considered.
Lung: reactive change consisting of moderate congestion, slight alveolar edema and minimal alveolar macrophages was recorded. (Macroscopic findings: discolouration, dark red)

FINDINGS IN SACRIFIED ANIMALS
All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

OTHER FINDINGS (PARENTAL ANIMALS)
CORPORA LUTEA COUNT
Mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (14.9, 13.4, 13.8 and 14.2 in order of ascending dose level) and gave no indication of a test item-related effect.

DURATION OF GESTATION
The mean duration of gestation was unaffected by exposure to the test item. Mean duration of gestation was 21.4, 21.8, 21.5 and 21.6 d, in order of ascending dose level.

IMPLANTATION RATE AND POST-IMPLANTATION LOSS
No test item-related effects were noted on implantation rate and implantation loss.

The total post-implantation loss was significantly decreased (p< 0.05) in females treated with 1000 mg/kg/day of the test item when compared with the controls, which was considered to be incidental.

The mean numbers of implantations per litter were 13.6, 11.4, 13.1 and 13.1 in order of ascending dose level. The mean incidence of post-implantation loss as a percentage of total implantations was 16.0, 17.5, 13.9 and 8.5 %, in order of ascending dose level.

LITTER SIZE AT FIRST LITTER CHECK
No effects were noted on litter size at first litter check.

In dose groups 0, 100, 300 and 1000 mg/kg/ day the birth index was 84.0, 83.5, 86.1 and 91.5% and mean litter size at first litter check was 11.5, 9.6, 11.3 and 12.0, respectively.

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM
No effects on the postnatal loss between day 0 and 4 post partum were noted.

Mean postnatal loss was 1.6, 1.2, 1.6 and 9.6% in dose groups 0, 100, 300 and 1000 mg/kg/ day, respectively. Correspondingly, in dose group 1000 mg/kg/day, the viability index was statistically significantly decreased (90.4%). In does groups 0, 100 and 300 mg/kg/day, the viability index was 98.4, 98.8 and 98.4%.

The cause of the slightly higher postnatal loss in dose group 1000 mg/kg/day was the loss of 7 pups on day 2 and 3 post partum for dam no. 84. For this dam already 6 dead pups at first litter check were noted. This isolated occurrence was considered to be incidental.
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 effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
CLINICAL SIGNS (OFFSPRING)
No abnormal findings were noted at first litter check or during the first 4 days post partum.

BODY WEIGHT (OFFSPRING)
Mean pup weights on day 1 and day 4 post partum were unaffected by treatment of their dams with the test item.

The mean difference in pup weight in the 1000 mg/kg/day treatment group, was slightly decreased (P< 0.05) when compared male and female pups of this group with the control group. Since the difference was not statistically significant, it was considered to be not test item-related.

GROSS PATHOLOGY (OFFSPRING)
No test item-related macroscopic findings were noted in pups at necropsy.
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 test-item related findings were noted at first litter check and during lactation in pups at any dose level
Critical effects observed:
no
Reproductive effects observed:
no

Summary of performance

P animals breeding for F1 litters

Group

(mg/kg/day)

1

(0)

2

(100)

3

(300)

4

(1000)

Female numbers

45 - 55

56 – 66

67 – 77

78 – 88

Number of females paired (A)

11

11

11

10

Number of females mated

11

10

11

10

Number of females, which died before the scheduled necropsy (B)

0

0

0

4

Number of females not pregnant (C)

0

1

0

0

Number of females which reared their pups until day 4post partum

11

9

11

7

(A) Female no. 88 died spontaneously during pregnancy

(B)Females died spontaneously, no.88 during pre-pairing, 86 during gestation, 83/87 during lactation

(C)Female no. 63 was not pregnant

Conclusions:
The reproductive toxicity via the oral route of the test item was assessed according to OECD Guideline 422 at dose levels of 100, 300 and 1000 mg/kg/day. Based on the results of the study, a NOAEL for general toxicity in males and females was considered to be 1000 mg/kg/day, the highest dose level tested.
Executive summary:

A 28 day repeat dose oral toxicity study combined with a reproductive/ developmental toxicity screening test was performed in the rat in accordance with OECD Guideline 422. The test item was administered by gavage to 3 groups, each of 11 male and 11 female RCCHan™:WIST strain rats for up to 8 weeks (including a 2 week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. There were no treatment related effects observed on mating, fertility or gestation length at any dose level. The offspring litter size, sex ratio, viability, growth and development were al comparable to controls and no adverse effects were noted. Since no treatment-related effects were observed for reproduction, a NOAEL was considered to be 1000 mg/kg bw/day. Furthermore, the study showed that the administration of the test item over a period of 28 days did not results in any toxicologically significant effects and hence the NOAEL for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day.

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

Effects on developmental toxicity

Description of key information

Embryo-Fetal Toxicity Study in Rats via Oral Gavage (OECD 414):

The purpose of this study was to assess any gross maternal and/or embryo-fetal toxicity of Cis‑hex-3-en-1-ol (LFA [Leaf Alcohol]) in the rat, following daily oral gavage administration during the period of major organogenesis, from implantation to the approximate day of closure of the hard palate, Gestation Days (GD) 6 to 19, with Cesarean section on GD 20.

Time-mated femaleSprague-Dawley CD®rats(20/group) were gavaged once daily with 0 (Polyethylene glycol 300, NF), 100, 300 or 1000 mg/kg/dayCis-hex-3-en-1-ol (LFA [Leaf Alcohol])from Gestation Days (GD) 6 to 19, inclusive (the period of major organogenesis, from implantation to the approximate day of closure of the hard palate). The dose volume was 5 mL/kg/day for all dose groups. All animals were euthanized on GD 20 with Cesarean sections and necropsies performed. Parameters evaluated during the study were: viability, clinical observations, body weights and food consumption. A macroscopic postmortem evaluation was performed on all animals on GD 20, during which counts of corpora lutea and implantations and uterine weights were recorded; fetuses were removed, weighed (live fetuses only), sexed and examined externally for defects,for soft tissue abnormalities and for skeletal abnormalities and ossification state. Placentas were examined and weighed. 

There were no Cis-hex-3-en-1-ol (LFA [Leaf Alcohol]) treatment-related deaths at ≤1000 mg/kg/day during this study. There were no test item-related clinical observations, body weight or body weight gains, and food consumption changes at ≤1000 mg/kg/day LFA. There were no test item-related effects on pregnancy or cesarean section parameters, macroscopic observations, gravid uterine weights, litter weights, fetal and placental weights, or fetal external, visceral and skeletal development.

Based on the no adverse effects observed at ≤1000 mg/kg/day LFA, the maternal and the developmental No-Observed-Adverse-Effect-Level (NOAEL) was determined to be 1000 mg/kg/day, when administered orally daily from GD 6 to GD 19. There was no teratogenic potential of Cis-hex-3-en-1-ol (LFA [Leaf Alcohol]) at≤1000 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Remarks:
pre-natal development toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study initiation: 4 October 2019; Study completion: 26 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
yes
Remarks:
Minor deviations from stuy plan but not considered to comprimise the validity or integrity of the study (see overall remarks)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Number of Animals:
The number of animals (20 time-mated females per group) was considered the minimum necessary to allow for meaningful interpretation of the outcome. The justification for 20 time mated females per group was based on the following two key concepts:
•The OECD Test Guideline 414 states that groups should contain a sufficient number of females to result in approximately 20 female animals with implantation sites at necropsy. Groups with fewer than 16 animals with implantation sites may be inappropriate. Some of the endpoints of principal interest (e.g., embryonic death, malformation) are low-frequency events .
•The normally expected pregnancy rate in this strain from the current vendor is approximately 90% with a range of 59 to 100%

Animal Information:
Animal model : Albino Rats (Outbred) VAF/Plus CD (Sprague-Dawley derived) [Crl:CD (SD]
Supplier: Charles River Laboratories
Number of animals received/placed on test: 80 time-mated females. The female rats selected for breeding were nulliparous and preferably virgin.
Age of the animals at receipt: Approximately 10 to 12 weeks on Gestation Day (GD) 0, where GD 0 was the day of detection of a copulatory plug in situ and/or sperm in a vaginal smear.
Weight range of the animals at start of treatment (GD 6): 227 g to 299 g

Pretest Period:
A 2 to 4 day pretest period was completed prior to commencement of dosing as dictated by the mating and receipt schedule of the animals. All animals were examined during the pretest period to confirm suitability for study. During this period, routine husbandry care and identification procedures were performed.

Assignment: Animals were randomly allocated to control or treated groups on arrival. Prior to dosing, the body weights obtained on GD 4 were reviewed to ensure minimization of inter-group variability based on those body weights.

Environmental Control:
Light/dark cycle: A twelve hour light/dark cycle was provided and controlled via an automatic timer.
Temperature and relative humidity: Monitored and maintained within the range of 20 to 26 °C and 30 to 70%.
There were no deviations from these ranges.

Animal Housing and Environmental Enrichment:
Cages: Polycarbonate cages with a stainless steel mesh lid.
Number of animals per cage: Individually housed.
Bedding: Teklad 7070C Certified Diamond Dry Cellulose Bedding. Provided to each cage throughout the study and changed at appropriate intervals each week.
Environmental enrichment: Provided to each cage throughout the study and replaced when necessary.

Feed:
Diet: Teklad Global 18% Protein Rodent Diet (Certified), 2018C
Availability: Without restriction. Fresh feed was presented as required.

Water:
Suppy: Potable water from the public supply via an automated watering system.
Availability: Without restriction

























Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
(Polyethylene glycol 300, NF (PEG 300))
Details on exposure:
TEST ITEM PREPARATION:
Preparation of Dose Formulations:
Cis-hex-3-en-1-ol (LFA [Leaf Alcohol]) was prepared for administration by adding the appropriate amount of vehicle to the test item to achieve the desired concentrations and mixed with a magnetic stirrer.

Group 1: Control (Dose:0 mg/kg/day; Concentration: 0 mg/mL; Volume dose: 5 mL/kg)
Group 2: LFA (Dose: 100 mg/kg/day; Concentration: 20 mg/mL; Volume dose: 5 mL/kg)
Group 3: LFA (Dose: 300 mg/kg/day; Concentration: 60 mg/mL; Volume dose: 5 mL/kg)
Group 4: Test iem (Dose: 1000 mg/kg/day; concentration: 200 mg/mL; Volume dose: 5 mL/kg)

The test item was used as supplied when calculating quantities to be used during dose preparation. Fresh formulations were prepared weekly and were stored at room temperature (15-25 °C) when not in use.

Detailed records of test item usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined at each preparation.

ADMINISTRATION OF TEST AND/OR CONTROL ITEM:
Route: Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg/day.
Volume dose : 5 mL/kg body weight.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Control (Group 1): Vehicle at the same volume dose as the treated groups.
Frequency and duration: The test and control (vehicle) items were administered once daily at approximately the same time each day (± 1 hour), 7 days per week, from GD 6 to 19, inclusive, where GD 0 is the day of detection of mating (copulatory plug in situ and/or sperm in a vaginal smear).
Sequence: By group
Formulation: Formulations were stirred using a magnetic stirrer for at least 20 minutes prior to and throughout the dosing procedure.
A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations of the test item in vehicle, Polyethylene glycol 300, NF (PEG 300), were analyzed to confirm that the prepared dose forumulations were homogenious and that the administered concentrations were appropriate under the study conditions.

The analytical method validated involved dilution of test item formulation samples with sample diluent (100% Acetone) followed by quantification using gas chromatography with flame ionization detection (GC-FID).

The homogeneity and concentration results for the test item dose formulations of all groups analysed during the course of the study met the study plan specified acceptance critera. No test item was detected in the control samples. Therefore, all dose formulations used for dosing in this study met teh acceptance criteria required by the study plan.

Details on mating procedure:
- Proof of pregnancy: detection of a copulatory plug in situ and/or sperm in a vaginal smear (Gestation Day 0)
Duration of treatment / exposure:
The test and control (vehicle) items were administered once daily at approximately the same time each day (± 1 hour), 7 days per week, from GD 6 to 19, inclusive, where GD 0 is the day of detection of mating.
Frequency of treatment:
Once daily
Duration of test:
Gestation Day 0 (mating) to Gestation Day 20 (Cesarean section).
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 - control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
20 females per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
The purpose of this study was to assess any gross maternal and/or embryo-fetal toxicity of Cis hex-3-en-1-ol (LFA [Leaf Alcohol]) in the rat, following daily oral gavage administration during the period of major organogenesis, from implantation to the approximate day of closure of the hard palate, Gestation Days (GD) 6 to 19, with Cesarean section on GD 20.

- Dose selection rationale:
In an OECD 422 study (D51351) Cis-hex-3-en-1-ol (leaf alcohol) was administered orally (gavage) to female rats at dose levels of 0 (PEG 300), 100, 300 and 1000 mg/kg/day (dose volume was 5 mL/kg) for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached Day 3 or 4 postpartum (a maximum of 54 consecutive days). There were no test item-related effects on mortality, clinical observations, body weights, food consumption and functional observational battery at doses ≤ 300 mg/kg/day. In females at 1000 mg/kg/day, mean body weight, body weight gain and food consumption were slightly reduced (non-adverse) when compared with the control group. The mean precoital time, fertility index, gestation length, corpora lutea count, pre- and post-implantation loss, litter size at first litter check and postnatal loss were not affected by the treatment at ≤ 1000 mg/kg/day. There were no test item-related changes in the mean organ weights, organ to body weight ratios and organ to brain weight ratios and no gross lesions observed at necropsy in females treated with ≤ 1000 mg/kg/day. Based on the OECD 422 results, a NOAEL (No Observed Adverse Effect Level) for general toxicity in females was considered to be 1000 mg/kg/day, the highest dose level tested.
Maternal examinations:
DAILY OBSERVATIONS:
Animals were observed in their cages twice daily for mortality and general condition.

DOSING OBSERVATIONS:
During the treatment period, all animals were observed for signs of toxic or pharmacologic effects once daily (1 ± 0.5 hour) after test item administration was completed for all animals in their respective dose groups.

SIGNS:
Animals were removed from their cages and examined on GD 4, 6, 9, 12, 15, 18 and at termination. During the stabilization period and on GD 20, these evaluations were performed in the morning. During the treatment period, these evaluations were performed prior to first daily dosing (within approximately 30 minutes). Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as evaluations of respiration.

BODY WEIGHT:
Animals were weighed on GD 4 and daily from GD 6 to 20.

FOOD CONSUMPTION:
Food consumption was measured (weighed) on GD 4 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 20.

TERMINAL INVESTIGATIONS:
MATERNAL SACRIFICE:
Macroscopic Examinations:
A necropsy (macroscopic examination) comprised of an external examination followed by an internal examination (excluding the cranial cavity) was performed. Minor sporadic lesions common to this strain of animal (i.e. localized patchy hair loss) were not sampled.

Scheduled Necropsy:
Scheduled necropsy was on GD 20.

Method of Euthanasia:
All adult females were euthanized by carbon dioxide inhalation followed by exsanguination. Live fetuses were euthanized by intraperitoneal injection of sodium pentobarbital.















Ovaries and uterine content:
Maternal sacrifice:
The intact uterus (ovaries attached) was removed from the abdominal cavity and weighed for all animals. Corpora lutea were counted and the number per ovary recorded for all animals. The uterus was then opened and the number and location of the following were recorded for each uterine horn:

- live fetuses
- dead fetuses (recently dead, no significant degeneration)
- late embryo-fetal deaths (recognizable dead embryo/fetus undergoing degeneration, regardless of size)
- early embryonic deaths (evidence of implantation, but no recognizable embryo), or stain-positive (see below)

The placenta associated with each fetus was examined macroscopically and weighed.

For apparently non-pregnant animals, and for apparently empty uterine horns, the number of implantation sites was checked by ammonium sulfide staining of the uterus (method modified from Salewski (Salewski, 1964)).
Fetal examinations:
EXTERNAL EXAMINATIONS:
All live fetuses at the scheduled necropsy were weighed, sexed, individually identified and examined externally for abnormalities, including examination of the eyes and palate.
All resorptions were discarded.

SOFT TISSUE (VISCERAL) EXAMINATIONS:
Following external examination, approximately one-half of the live fetuses in each litter (nominally alternating fetuses within the uterus) were euthanized and placed in Bouin’s fixative for preservation and decalcification. These fetuses were examined for soft tissue abnormalities by means of a combination of a micro-dissection technique derived from that of Staples (Staples, 1974) for examination of the torso, and a free-hand slicing technique derived from that of Wilson and Warkany (Wilson & Warkany, 1965) for examination of head and heart structures. During the examination, the sex of each fetus was determined by inspection of the gonads.

SKELETAL EXAMINATION:
Following external examination, approximately one-half of the live fetuses in each litter (nominally, alternating fetuses within the uterus) were euthanized, sexed by internal inspection, then eviscerated and processed for staining of the skeleton with Alizarin Red S. These preparations were then examined for skeletal abnormalities, including any affecting discernible cartilaginous structures, and for ossification state.
Statistics:
All statistical analyses were carried out using the individual animal or litter as the basic experimental unit.
The following data types were analyzed at each timepoint separately:

- maternal body weight
- maternal cumulative body weight change from baseline
- food consumption
- fetal weights per litter
- placental weights per litter
- litter size
- count of corpora lutea
- count of implantation sites
- percent pre-implantation loss
- percent post-implantation loss (resorptions and early/fetal deaths)
- sex ratio per litter

The following comparisons were made: Group 1 (control) vs 2, 3 and 4.

Indices:
Maternal sacrifice:
Gravid uterine adjusted body weight was calculated as:
Adj Bwt Day 20 = Body weight on Day 20 – Gravid Uterine Weight

Gravid uterine adjusted body weight change was calculated as:
Adj bwt change Days 6-20 = Adjusted body weight Day 20 – Body weight on Day 6

Pre-implantation loss was calculated as:
Number of corpora lutea - Number of implantations

Percent pre-implantation loss was calculated as:
(Number of corpora lutea - Number of implantations) ÷ Number of corpora lutea x 100

Post-implantation loss was calculated as:
Total number of resorptions + number of dead fetuses

Percent post-implantation loss was calculated as:
(Total number of resorptions and dead fetuses) ÷ Number of implantation sites x 100

Sex ratio (% males) was calculated as:
(Number of male pups ÷ Number of total pups) x 100







Historical control data:
Fetal examination results compared to published historical control ranges:
References:
Lewis, 2013. Reproductive Toxicology Historical Control Data for the Crl:CD® BR Rat 2008 to 2012. (2013). Elise M. Lewis, PhD, Director of Reproductive and Neurobehavioral Toxicology, Charles River Laboratories, Preclinical Services, Pennsylvania.

Lewis, 2016. Reproductive Toxicology Historical Control Data for the Crl:CD® BR Rat 2011 to 2015. (2016). Elise M. Lewis, PhD, Director of Reproductive and Neurobehavioral Toxicology, Charles River Laboratories, Preclinical Services, Pennsylvania.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test item-related clinical observations at ≤1000 mg/kg/day LFA.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no deaths at ≤1000 mg/kg/day LFA.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Gestation Body Weights:
There were no test item-related adverse effects on body weights and body weight gains at ≤1000 mg/kg/day LFA.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item-related adverse effects on food consumption at ≤1000 mg/kg/day LFA.
Food efficiency:
no effects observed
Description (incidence and severity):
See food consumption.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related adverse effects on macroscopic evaluations at ≤1000 mg/kg/day LFA.
The only macroscopic finding (abnormal uterine/cervical color in a single control group animal) in this study was considered not test item-related because it occurred in the control group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
All pregnant females had viable fetuses at Cesarean sectioning.

See Pregnancy Outcome and Cesarean Section Data plus summary of results tables.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
implantations, % pre-implantation and % post-implantation losses were comparable to mean control values.

See Pregnancy Outcome and Cesarean Section Data plus summary of results tables.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
See Pregnancy Outcome and Cesarean Section Data.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Resorptions (early, late and total) were comparable to mean control values.

See Pregnancy Outcome and Cesarean Section Data plus summary of results tables.
Dead fetuses:
no effects observed
Description (incidence and severity):
Dead fetuses were comparable to mean control values. No dead fetuses.

See Pregnancy Outcome and Cesarean Section Data plus summary of results tables.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
See Pregnancy Outcome and Cesarean Section Data.

3 non-pregnant animals in control and 100 mg/kg/day groups. 2 non-pregnant animals in 300 mg/kg/day group. 4 non-pregnant animals in 1000 mg/kg/day group.

Reason for non-pregnancy not specified but not considered related to treatment as non-pregnant animals in control group and no dose-response relationship.
Details on maternal toxic effects:
Pregnancy Outcome and Cesarean Section Data:
There were no test item-related adverse effects on pregnancy outcome and Cesarean section data at ≤1000 mg/kg/day LFA.

There were 17, 17, 18 and 16 pregnant females in the 0, 100, 300 and 1000 mg/kg/day groups, respectively. All pregnant females had viable fetuses at Cesarean sectioning. There were statistically significant increases in corpora lutea in the 300 and 1000 mg/kg/day LFA groups. The number of corpora lutea were determined prior to treatment and therefore are not toxicologically relevant. All other Cesarean section parameters (sex ratio, implantations, resorptions [early, late and total], dead fetuses, live fetuses [male, female and total], sex ratio (% Male), and % pre implantation and % post implantation losses) were comparable to mean control values.




Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no test item-related clinical observations, body weight or body weight gains, and food consumption changes at ≤1000 mg/kg/day LFA. There were no test item-related effects on pregnancy or cesarean section parameters, macroscopic observations.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no test item-related effects on gravid uterine weights,and fetal and placental weights at ≤1000 mg/kg/day LFA.

See summary of results tables.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Live fetuses [male, female and total] were comparable to mean control values.

See summary of results tables.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio (% Male) were comparable to mean control values:
Control: 45.6%
100 mg/kg/day: 50.5%
300 mg/kg/day: 49.9%
1000 mg/kg/day: 51.0%
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no test item-related effects on litter weights at ≤1000 mg/kg/day LFA.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related fetal external abnormalities at ≤1000 mg/kg/day LFA.

Major external findings (thoracogastroschisis, iniencephaly, ablepharia, brachygnathie, forepaw flecture protruding tongues [fetus 1511-6]) were not considered related to the test item as these findings were observed in a single fetus and single litter in the control group. Minor external findings (kinked tail [fetuses 1516-10, 1517-7, 3543-13, 4565-6, -10, -13, -15 and 4574-6 from 2, 0, 1 and 2 litters in the 0, 100, 300 and 1000 mg/kg/day LFA groups, respectively] and tail tip with fleshy tab [3542-2]) were not considered related to the test item as they were low in litter incidence, were similar with the concurrent control value, and/or were within published historical control ranges.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related fetal skeletal abnormalities at ≤1000 mg/kg/day LFA.

Minor skeletal findings (e.g., cervical ribs, misshapen scapula, thickened and/or short ribs, rudimentary 14th ribs) were observed in the treated groups but were not considered test item related because the findings were low in incidence, not present in a dose-related manner, were within internal and/or published historical control data ranges and/or were common in this strain of laboratory animal.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related fetal visceral abnormalities at ≤1000 mg/kg/day LFA.

There were no major visceral finings. Minor visceral findings (malpositioned epididymis, malpositioned testis, subdural hemorrhage in the cerebellar and cerebral hemispheres, subdural hemorrhages in the midbrain, olfactory lobe and region, hemorrhage in the epidural space of the medulla oblongata, anomalous confluence of the caudal vena cava and left hepatic vein, innominate absent or short, pulmonary trunk dilated, subclavian artery arising from aortic arch, partially undescended thymus gland, small renal papilla, dilated renal pelvis, dilated ureter and additional median liver lobes) were not considered related to the test item as they were low in incidence, not present in a dose-related manner, occurred in the control group only, were within published historical control data and/or were common findings in this strain of laboratory animal.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Fetal Observations::
There were no test item-related effects on fetal external, visceral and skeletal development at ≤1000 mg/kg/day LFA.
There were 218, 224, 244 and 228 fetuses from 17, 17, 18 and 16 litters from the 0, 100, 300 and 1000 mg/kg/day groups, respectively, examined for external findings. Of these, 111, 113, 123 and 113 fetuses from the 0, 100, 300 and 1000 mg/kg/day groups, respectively, were examined for visceral findings and 107, 111, 121 and 115 fetuses from 17, 17, 18 and 16 litters, respectively, were examined for skeletal findings.
Details on embryotoxic / teratogenic effects:
There was no teratogenic potential of Cis-hex-3-en-1-ol (LFA [Leaf Alcohol]) at ≤1000 mg/kg/day.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no test item-related effects on gravid uterine weights, litter weights, fetal and placental weights, or fetal external, visceral and skeletal development.
Key result
Abnormalities:
effects observed, non-treatment-related
Key result
Developmental effects observed:
no

Full results tables (for group data) and appendixes (for individual animal data) are attached as background material.

Key data summarised below:

Number of pregnant and non-pregnant dams:

(for full data see Table 7 in attached background material).

 

Dose (mg/kgday)

0

100

300

1000

Number of females on study (mated)

20

20

20

20

Non-pregnant

3

3

2

4

Pregnant

17

17

18

16

% Pregnant

85

85

90

80

Pregnant with termination before scheduled date

0

0

0

0

Pregnant at scheduled termination – with total implantation loss

0

0

0

0

Pregnant at schedule termination – with viable fetuses

17

17

18

16

Reason for non-pregnancy not specified but within expected pregnancy rate range for strain of rat from supplier (approximately 90% with a range of 59 to 100%).

Number of dams with abortions, early-deliveries, stillbirths, resorptions, and/or dead foetuses:

(for full data see Table 8 and Appendix 8 in attached background material).

Dose (mg/kg/day)

0

100

300

1000

Abortions

0

0

0

0

Early deliveries

0

0

0

0

Still births

0

0

0

0

Resorptions*

0.6

0.9

0.9

0.9

Dead foetuses

0

0

0

0

*resorption figures based group mean values for total resorptions (early and late)

Pre- and post-implantation loss, number and percent:

(for full data see Table 8 and Appendix 8 in attached background material).

Dose (mg/kg/bw)

0

100

300

1000

Number pre-implantation loss**

242 – 228 = 14

262 – 240 = 22

291 – 261 = 30

255 – 242 = 13

% pre-implantation loss*

6.5%

8.7%

10.1%

5.8%

Number post-implantation loss***

10 + 0 = 10

16 + 0 = 16

17 + 0 = 17

14 + 0 = 14

% post implantation loss*

6.3%

6.7%

6.6%

5.4%

*% figures based on group mean values

** calculated as group total corpora lutea – group total implantations

***calculated as group total number of resportions + number of dead foetuses

Body weight, body weight change and gravid uterine weight, including optionally, body weight change corrected for gravid uterine weight:

(for full data see Table 9 and Appendix 9).

Group Mean Values (in g)

Dose (mg/kg/bw)

Bodyweight on Day 6

Bodyweight on Day 20

Bwt change Days 6-20

Gravid Uterine Weight

Adj Bwt Day 20

Adj Bwt change Days 6-20

0

267

395

128

81.37

313

47

100

274

407

133

84.48

323

49

300

265

399

134

85.88

313

48

1000

272

412

140

90.41

321

50

Mean number and percent of live offspring:

(see Table 8 in attached background material).

Dose (mg/kg/bw)

0

100

300

1000

Mean number live offspring*

12.8

13.2

13.6

14.3

% live offspring**

93.7

93.3

93.4

94.6

 

*based on group mean values

** calculated as 100% - % post-implantation loss

Mean foeatal/pup bodyweight by sex and sexes combined

(for full data see Table 10 and Appendix 10, 11, 12 in attached background material).

Group mean values (g)

Dose (mg/kg/bw)

Placental Weight

Litter Weight

Male Fetal Weight

Female Fetal Weight

Overall Fetal Weight

0

0.6

51.8

4.1

4.0

4.0

100

0.6

54.1

4.2

4.0

4.2

300

0.6

55.3

4.2

4.0

4.1

1000

0.6

57.2

4.1

3.9

4.0

Number and percent of foetuses and litters with malformations (including runts) and/or variation as well as description and incidences of malformations and main variations (and/or retardation).

 

Fetal External Observations:

(For full data see Table 11 and Appendix 13 in attached background material)

Dose (mg/kg/bw)

Number of fetuses examined

Number of litters examined

Test-item related abnormalities

Minor findings not considered related to test item

Description and incidences

0

218

17

No

Yes

See Table and Appendix

100

224

17

No

Yes

See Tables

and Appendix

300

244

18

No

Yes

See Table and Appendix

1000

228

16

No

Yes

See Table and Appendix

 

Fetal Visceral Observations:

(For full data see Table 12 and Appendix 13 in attached background material)

Dose (mg/kg/bw)

Number of fetuses examined

Number of litters examined

Test-item related abnormalities

Minor findings not considered related to test item

Description and incidences

0

111

17

No

Yes

See Table and Appendix

100

113

17

No

Yes

See Tables

and Appendix

300

123

18

No

Yes

See Table and Appendix

1000

113

16

No

Yes

See Tables and Appendix

 

Fetal Skeletal Observations:

(For full data see Table 13 and Appendix 13 in attached background material)

Dose (mg/kg/bw)

Number of fetuses examined

Number of litters examined

Test-item related abnormalities

Minor findings not considered related to test item

Description and incidences

0

107

17

No

Yes

See Table and Appendix

100

111

17

No

Yes

See Tables

and Appendix

300

121

18

No

Yes

See Table and Appendix

1000

115

16

No

Yes

See Tables and Appendix

Historical control data:

Fetal observations were compared against published historical control ranges and found to be within these ranges. See the published historical control data in attached background material.

Minor fetal external, visceral and skeletal observations observed were within the historical control data ranges and therefore not considered to be adverse effects related to the test item.

Conclusions:
There were no Cis-hex-3-en-1-ol (LFA [Leaf Alcohol]) treatment-related deaths at ≤1000 mg/kg/day during this study. There were no test item-related clinical observations, body weight or body weight gains, and food consumption changes at ≤1000 mg/kg/day LFA. There were no test item-related effects on pregnancy or cesarean section parameters, macroscopic observations, gravid uterine weights, litter weights, fetal and placental weights, or fetal external, visceral and skeletal development.

Based on the no adverse effects observed at ≤1000 mg/kg/day LFA, the maternal and the developmental No-Observed-Adverse-Effect-Level (NOAEL) was determined to be 1000 mg/kg/day, when administered orally daily from GD 6 to GD 19. There was no teratogenic potential of Cis-hex-3-en-1-ol (LFA [Leaf Alcohol]) at ≤1000 mg/kg/day.
Executive summary:

Embryo-Fetal Toxicity Study in Rats via Oral Gavage:

Time-mated femaleSprague-Dawley CD®rats (20/group) were gavaged once daily with 0 (Polyethylene glycol 300, NF), 100, 300 or 1000 mg/kg/day Cis-hex-3-en-1-ol (LFA [Leaf Alcohol]) from Gestation Days (GD) 6 to 19, inclusive (the period of major organogenesis, from implantation to the approximate day of closure of the hard palate). The dose volume was 5 mL/kg/day for all dose groups. All animals were euthanized on GD 20 with Cesarean sections and necropsies performed. Parameters evaluated during the study were: viability, clinical observations, body weights and food consumption. A macroscopic postmortem evaluation was performed on all animals on GD 20, during which counts of corpora lutea and implantations and uterine weights were recorded; fetuses were removed, weighed (live fetuses only), sexed and examined externally for defects, for soft tissue abnormalities and for skeletal abnormalities and ossification state. Placentas were examined and weighed. 

There were no Cis-hex-3-en-1-ol (LFA [Leaf Alcohol]) treatment-related deaths at ≤1000 mg/kg/dayduring this study. There were no test item-related clinical observations, body weight or body weight gains, and food consumption changes at ≤1000 mg/kg/day LFA. There were no test item-related effects on pregnancy or cesarean section parameters, macroscopic observations, gravid uterine weights, litter weights, fetal and placental weights, or fetal external, visceral and skeletal development.

Based on the no adverse effects observed at ≤1000 mg/kg/day LFA, the maternal and the developmental No-Observed-Adverse-Effect-Level (NOAEL) was determined to be 1000 mg/kg/day, when administered orally daily from GD 6 to GD 19. There was no teratogenic potential of Cis-hex-3-en-1-ol (LFA [Leaf Alcohol]) at ≤1000 mg/kg/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
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

In addition to the results of the OECD 414 study, the results from the OECD 422 study showed no test item-related findings at first litter check and during lactation in pups at any dose level.

Justification for classification or non-classification

The available combined repeatad dose toxicity study with reproduction/developmental toxicity test (OECD 422), administered via the oral route, has been assigned reliability 1 and is considered as acceptable for classification. The study showed that the administration of the test item over a period of 28 days did not results in any toxicologically significant effects and hence the NOAEL for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day. As such, the test item can be considered to be non-classified for reproductive toxicity.

The embryo-fetal toxicity study in rats via oral gavage (pre-natal developmental toxicity) (OECD 414) no adverse effects at any dose level (≤1000 mg/kg/day LFA). The NOAEL was determined to be 1000 mg/kg/day and no teratogenic potential at ≤1000 mg/kg/day LFA. As such, the test item is not classified for developmental toxicity.