Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

 This endpoint was fulfilled using read across from Geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol) in a dermal study and from a reaction mass of Nerol (cis-3,7-dimethyl-2,6-octadien-1-ol ) and Geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol) in an oral study, for which the following results were obtained.

A dermal reproductive toxicity study was conducted according to OECD guideline 421 at concentrations 50, 150, 300 and 450 mg/kg bw/day of Geraniol. Due to severe dermal findings, the dose level for test group 3 was decreased to 300 mg/kg bw/d from study day 10 onwards. Regarding clinical examinations, only signs of local dermal toxicity were observed for males and females at all dose levels. There were no signs of systemic toxicity. The no-observable-adverse-effect-level (NOAEL) for systemic toxicity of the test item is 300 mg/kg/day. There was no treatment related effect on reproduction or development of the pups and therefore the reproductive and F1 NOAEL was also 300 mg/kg bw/day.

An oral reproductive toxicity study was conducted according to OECD guideline 421 at concentrations 100, 300 and 1000 mg/kg bw/day Nerol/Geraniol.

Clinical observations indicated distinct toxicity in the exposed parental animals of the highdose group (1000 mg/kg bw/d) but not in the animals of the mid- and low-dose group.

A reduction of food consumption (up to 10% in males and females during treatment weeks 0-1 as well as females (-34%) during lactation), and decreased body weight in males (up to -5%) had been determined during treatment weeks 2-4. A similar pattern as for clinical observations was noted for body weight and body weight change of the parental animals. A distinct decrease was noted in the high-dose animals of both sex even manifested in different time periods of the study. The body weight change in males was reduced from week 0 to 5 (-28% on average in this time period). In females a significant body weight change was observed during lactation leading to a body weight loss (-3%). Consequently, the body weight was decreased in males during treatment weeks 2-5 (-7%) and in females of week 6 (-9%). Therefore the NOAEL for systemic toxicity in adults was 300 mg/kg bw/day.

The test compound did not adversely affect fertility of the F0 generation parental animals at all dose levels as there were no changes of male/female mating and fertility indices, time until successful copulation, duration of pregnancy and mean number of implantations. Therefore, the NOAEL for fertility was 1000 mg/kg bw/day.

However, there is an alert for a dose-dependent adverse effect of the test substance on pre-/postnatal development of the F1 offspring at mid and high-dose level (300 and 1000 mg/kg bw/d). At the high dose level, this was indicated by a decreased number of delivered/liveborn and an increased number of stillborn pups, resulting in a distinctly reduced pup survival (– 25%). This reduced live birth index was due to losses in only one animal in the group (all others showed no losses). The significantly reduced postnatal offspring weight/weight gain during the first 4 days after birth are likely related to maternal toxicity and ability to care and nurse for the pups as evidenced by clinical observations, empty stomachs in 10% of pups and significantly reduced feed consumption and body weights during the lactation period. Overall, it may be considered that the pup effects seen are secondary to maternal toxicity which effects pup care and nursing.

At the mid dose level, the same effects were noted, but at a lesser incidence and no significant effect on weight/weight gain was observed. The slightly higher (non-significant) number of stillborns may well be contributed to the greater litter size in this group, which leaves the adverse effects on development of offspring in the mid-dose group to be limited to a slightly reduced pup survival (-9%). At least partially, the reduced pup survival may be secondary to a disturbance of maternal care as it became obvious by empty stomachs in pups which have been observed in 5% of mid-dose and 10% of high-dose offspring. In addition, findings in the mid dose appear to be limited to one animal. This animal appears to have maternal toxicity issues as evidenced by clinical observations (pups not properly nursed and insufficient maternal care of the pups), significantly reduced feed consumption and empty stomachs in the pups. These effects are similar to those seen at the top dose and it may therefore be concluded that maternal toxicity in this one animal is also responsible for the effects seen. The NOAEL for the F1 generation was therefore 100 mg/kg bw day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: guideline study acc. to GLP
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
dermal application
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Germany
- Age at study initiation: about 11-12 weeks
- Housing: Makrolon cage type M III
- No. of animals per cage: 1 animal,
- Exceptions: during mating: 1 male/ 1 female per cage; during rearing up to PND4: 1 dam with her litter
- Enrichment: Wooden gnawing blocks (Type NGM E-022)
- Bedding: Type Lignocel PS 14 fibres, dustfree bedding
- Diet: Ground Kliba maintenance diet mouse/rat "GLP"; ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12:12
Route of administration:
dermal
Vehicle:
corn oil
Details on exposure:
- Preparation frequency: The preparations were prepared at intervals for which the stability is guaranteed (7 days).
- Application area: Intact clipped skin of the back (dorsal and dorsolateral areas of the trunk; not less than 10% of the body surface); the first clipping was carried out at least 24 hours before the randomization. The rats were reclipped at least once a week (depending on the hair growth).
- Type of application: Dermal application of the test-substance preparations to the clipped intact dorsal skin by means was carried out with 3-mL syringes (3CC Syringe, supplied by Becton, Dickinson & Co., Franklin Lakes, U.S.A.) and a semiocclusive dressing (4 layers of absorbent gauze) and stretch bandage)). The test-substance preparation was applied to the dorsal skin with the syringe in each case. After removal of the dressing, the application area was washed with lukewarm water.
- Volume to be applied: 4 mL/kg body weight (related to the body weight determined most recently in each case)
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up tp 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Application period:
Males: From day 0 (start of administration period) until sacrifice
Females: From day 0 (start of administration period) until GD 19
Frequency of treatment:
daily for at least 6 hours
Dose / conc.:
450 mg/kg bw/day
Remarks:
Doses / Concentrations:
450 mg/kg bw/day
Basis:
nominal conc.
initial high dose
Dose / conc.:
300 mg/kg bw/day
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
nominal conc.
reduced high dose due to massive skin irritation after 10 days of application
Dose / conc.:
150 mg/kg bw/day
Remarks:
Doses / Concentrations:
150 mg/kg bw/day
Basis:
nominal conc.
mid dose
Dose / conc.:
50 mg/kg bw/day
Remarks:
Doses / Concentrations:
50 mg/kg bw/day
Basis:
nominal conc.
low dose
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

DETAILED CLINICAL OBSERVATIONS: Yes
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented for each animal.
The parturition and lactation behavior of the dams was generally evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver or umbilical cord not cut) were documented on an individual dam basis.
On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.
The day of parturition was considered to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day. Deviations from this procedure were on Saturdays, Sundays and public holidays.

BODY WEIGHT:
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning).
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female parental animals:
• During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
• Females without litter were weighed once a week.
• Females between PND 4 and sacrifice were weighed once a week.

FOOD CONSUMPTION:
Generally, food consumption was determined once a week (in a period of 7 days) for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
• Food consumption of the females, which gave birth to a litter, was determined for PND 1-4.
Food consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.
Litter observations:
Pup number and status at delivery
All pups delivered from the F0 parents were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn pups in each litter. At the same time, the pups were examined for gross-morphological changes. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.

Pup viability/ mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays.
The number and percentage of dead pups on the day of birth (PND 0) and pups dying during the lactation period were determined. However, pups which died accidentally and pups which were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day of birth and on PND 4. Furthermore, the viability index was calculated according to the following formula:
Viability index (%) = (number of live pups on day 4 after birth/number of liveborn pups on the day of birth)x 100

Sex ratio
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle. Normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups finally confirmed at necropsy.
The sex ratio was calculated at PND 0 and 4 according to the following formula:
Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4)x100

Pup clinical observations
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned.

Pup body weight data
The pups were weighed one day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results.
Furthermore the body weights on PND 1 were used for the calculation of "runts" (pups, which weighed less than 25% of the mean weight of the respective control pups). The individual weights were always determined at about the same time of the day (in the morning).
In the relevant summary tables pup body weights (including "runts") and pup body weight gains are listed for males, females as well as males and females together.
Postmortem examinations (parental animals):
Necropsy
All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Animals which have died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after their death and assessed by gross pathology.

Organ weights
Weight assessment was carried out on all animals sacrificed at scheduled dates. The following weights were determined:
1. Anesthetized animals
2. Epididymides
3. Testes
4. Ovaries

Organ/tissue fixation
The following organs or tissues of parental animals were fixed in 4% neutral buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Epididymides (fixed in modified Davidson’s solution)
4. Ovaries (fixed in modified Davidson’s solution)
5. Pituitary gland
6. Prostate gland, seminal vesicles, coagulation glands
7. Skin treated
8. Skin untreated
9. Testes (fixed in modified Davidson’s solution)
10. Uterus, oviducts, vagina
The liver, ovaries, testes and epididymides of animals that died or were sacrificed intercurrently were fixed in 4% buffered formaldehyde solution.

Histopathology
After the organs were fixed, histotechnical processing and examination by light microscopy were performed according to the following table:

Organs Test groups
0 1 2 3
All gross lesions A2 A2 A2 A2
Epididymides A1 A1
Ovaries A1 A1
Skin treated A1 A1 A1 A1
Skin untreated A1 A1
Testes A1 A1

A = hematoxylin and eosin stain
1 = all animals per group
2 = all animals affected per group
Animals that have died or were sacrificed in a moribund state were processed histotechnically and assessed like control animals.
Postmortem examinations (offspring):
Pup necropsy observations
All surviving pups (sacrificed on PND 4 under isoflurane anesthesia with CO2), all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
massive skin irritation at 450 mg/kg bw/d
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Lymphocytic infiltrates were observed in treated skin sections which were distributed in an interface pattern
Reproductive performance:
no effects observed
Clinical observations for males
For males, several dermal findings were noted in test groups 3 (450 and 300 mg/kg bw/d). Starting on study day 10, 5 rats showed focal and multifocal red spots on treated skin on several days. Starting on study day 11, 9 male rats showed focal scales on treated skin on several days. One rat showed focal erosion on treated skin on several days beginning on study day 14. Starting on study day 15, 5 rats showed slight erythema on treated skin onseveral days.
Similar findings but less pronounced were observed in male animals of test group 2 (150 mg/kg bw/d), i.e. focal red spots on treated skin on several days in 5 animals from study day 9 onwards, focal scales on treated skin starting on study day 13 in 2 animals and slight erythema on treated skin in 3 animals starting on study day 15.
No treatment-related findings were observed in male animals of test group 1 (50 mg/kg bw/d).

Clinical observations for females
As observed for the male animals of test group 3 (450 and 300 mg/kg bw/d) different dermal findings on treated skin were noted on several days, i.e. during premating, mating and lactation periods. Starting on study day 3, seven animals showed slight and moderate erythema on several days of the study. In addition, some animals of test group 3 (450 and 300 mg/kg bw/d) showed multifocal, focal and diffuse scales on treated skin starting on study day 5.
Similar findings but less pronounced were observed in female animals of test group 2 (150 mg/kg bw/d), i.e. focal red spots, slight erythema and focal scales on treated skin on several days of the study.
In test group 1 (50 mg/kg bw/d) slight erythema as well as focal and diffuse scales on treated skin were observed in individual animals at different time points.
One female of test group 3 (450 and 300 mg/kg bw/d), which did not deliver pups, showed a vaginal hemorrhage 27 days after mating.
In one female animal of test group 2 (150 mg/kg bw/d) a severe thoracal injury was observed during premating. It was a self inflicted injury which was caused by the animal’s attempt to get rid off the gauze. The injury became more severe by time and the animal had to be sacrificed in a moribund state in study week 1. A red-brown lesion was noted on the thorax of this animal, which correlated to an erosion/ulcer on the skin.

Histopathology
Lymphocytic infiltrates were observed in treated skin sections which were distributed in an interface pattern (10/10 males and 5/10 females at 450 mg/kg bw/d [test group 3] graded minimal to slight, 7/10 males at 150 mg/kg bw/d [test group 2] graded minimal, 2/10 males at 50 mg/kg bw/d [test group 1] graded minimal). All other findings noted were single observations which were considered to be incidental and spontaneous in origin and without any relation to treatment.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive performance and fertility
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Gross pathological findings:
no effects observed
Pup number and status at delivery
The mean number of delivered pups per dam and the rate of liveborn and stillborn pups were evenly distributed among test group 0, 1, 2 and 3 (0, 50, 150 and 450 and 300 mg/kg bw/d). The respective values reflect the normal range of biological variation inherent in the strain used in this study.

Pup viability/mortality
No significant findings for pups that died during lactation were observed. The viability index as indicator for pup mortality between PND 0-4 varied between 99% (test group 1, 50 mg/kg bw/d and test group 3, 450 and 300 mg/kg bw/d) and 100% (test group 0, 0 mg/kg bw/d and test group 2, 150 mg/kg bw/d). No test substance-related changes were obtained.
One male pup delivered by one dam of test group 1 (50 mg/kg bw/d) was sacrificed moribund on PND 0 as it showed malformation of the skull, anophthalmia and cleft lip (see Pup clinical observations).

Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show biologically relevant differences between the control and test groups 1-3.

Pup clinical observations
One dam of test group 1 (50 mg/kg bw/d) gave birth to a male pup with a deformation of its snout. On first sight a cleft lip and anophthalmia were seen. The staining of the pups skull showed that several bones (e.g. basisphenoid, palatine, incisive, nasal incl. cartilage and maxilla) were deformed and/or displaced.
Therefore, F1 pups did not show adverse clinical signs up to scheduled sacrifice on PND 4.

Pup body weight data
The mean body weight of female pups in test group 3 (450 and 300 mg/kg bw/d) was increased 11% on PND 1.
Pup body weight changes in the test substance-treated groups 1-3 (50, 150 and 450 and 300 mg/kg bw/d) were comparable to the concurrent control values.
The numbers of runts were 1 female in the control group and 1 male in test group 3 (450 and 300 mg/kg bw/d) on PND 1. Since a dose-response relationship was not observed a relation to dosing was not assumed.

Pup necropsy observations
During the necropsy of all F1 pups no test substance-related findings were observed.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Reproductive effects observed:
not specified
Conclusions:
The dermal administration of Geraniol Extra revealed only local signs of toxicity in male and female Wistar rats at all dose levels. This finding was related to the irritating potential of the test substance.
Thus, concerning toxicity to reproduction the test substance does not need to be classified neither according to Dir 1999/45/EC nor to Reg (EC) 1272/2008.
Executive summary:

Geraniol Extra was administered via dermal administration to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 (vehicle control; test group 0), 50 (test group 1), 150 (test group 2) and 450 mg/kg bw/d (test group 3) in order to observe the possible effects of the test substance on the integrity and performance of the reproductive system in both sexes. Due to severe dermal findings, the dose level for test group 3 was decreased to 300 mg/kg bw/d from study day 10 onwards.

Regarding clinical examinations, only signs of local dermal toxicity were observed for males and females at all dose levels. No changes in food consumption and body weight data were seen at any dose level.

Fertility indices for male and female animals were not impaired by test-substance administration.

Regarding pathology, there were no treatment-related necropsy or histological findings in ovaries, testes or epididymides associated with dermal administration of the test substance. The local minimal inflammatory reactions in the skin of treated males (test groups 1-3) and females (test group 3 only) were regarded as related to treatment and adverse.

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: guideline study acc. to GLP
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Germany
- Age at study initiation: about 9 weeks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:
- Housing: Makrolon cage type M III
- No. of animals per cage: 1 animal,
- Exceptions: during mating: 1 male/ 1 female per cage; during rearing up to PND4: 1 dam with her litter
- Enrichment: Wooden gnawing blocks (Type NGM E-022)
- Bedding: Type Lignocel PS 14 fibres, dustfree bedding
- Diet: Ground Kliba maintenance diet mouse/rat "GLP"; ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12:12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
dose volume: 4 ml/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up tp 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Males:
- 14 days premating
- up to 14 days mating
- sacrifice minimum 28 days after first application

Females:
- 14 days premating
- up to 14 days mating
- gestation about 22 days
- sacrifice minimum 4 days after littering
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
Mortality
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied. The examinations of these animals were carried out according to the methods established at the pathology laboratory.

Clinical observations
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal. For technical reasons, however, the clinical observations recorded during the premating period were printed out on a weekly basis. Individual data of daily observations can be found in the raw data.
The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.
On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.
The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

Food consumption
Generally, food consumption was determined once a week (in a period of 7 days) for male and female parental animals, with the following exceptions:
• Food consumption was not determined during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0, 7, 14 and 20.
• Food consumption of F0 females, which gave birth to a litter was determined on PND 1 and 4.
Food consumption was not determined in females without litter during lactation period.

Body weight data
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice.
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
• Females without litter, waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.
Litter observations:
Pup number and status at delivery
The status (sex, liveborn or stillborn) and number of all delivered pups were determined as soon as possible on the day of birth. At the same time, the pups were also examined for macroscopically evident changes. Pups that die before this initial examination are defined as stillborn pups.

Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods, which are described in detail in section 3.8.2.4. “Necropsy observations”.
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. The viability index was calculated according to the following formula:
Viability index (%) = (number of live pups on day 4 after birth/number of live pups on the day of birth) x100

Sex ratio
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy.
The sex ratio was calculated at day 0 and day 4 after birth according to the following formula:
Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) x100

Pup clinical observations
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned.

Pup body weight data
The pups were weighed on the day after birth (PND 1) and on PND 4.
Pups' body weight change was calculated from these results.
The individual weights were always determined at about the same time of the day (in the morning).
“Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.
Postmortem examinations (parental animals):
Necropsy
All animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Special attention was given to the reproductive organs.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Epididymides
3. Testes
4. Ovaries

Organ / Tissue fixation of parental animals
The following organs or tissues of parental animals were fixed in neutral buffered 4%
formaldehyde or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Pituitary gland
4. Testis (fixed in modified Davidson’s solution)
5. Epididymides (fixed in modified Davidson’s solution)
6. Prostate gland, seminal vesicles, coagulation glands
7. Ovaries (fixed in modified Davidson’s solution)
8. Uterus, oviducts, vagina
The uteri of all cohabited female F0 parental animals were examined for the presence and number of implantation sites.
The uteri of apparently nonpregnant animals or empty uterus horns were placed in 10% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method). Then the uteri were rinsed carefully under running water. When the examinations are completed, the uteri were transferred to the Pathology Laboratory for further processing.

Histopathology of parental animals
After the organs were fixed, histotechnical processing and examination by light microscopy was performed according to the following table:

Organs Test groups
0 1 2 3
1. All gross lesions A2 A2 A2 A2
2. Testes A1 A1
3. Epididymides A1 A1
4. Ovaries A1 A1

Methods and scope of examination:
A = hematoxylin and eosin stain
1 = all animals per group
2 = all animals affected per group

The hematoxylin-eosin stained slides were examined by light microscopy and assessed. A correlation between gross lesions and histopathological findings was performed.
Postmortem examinations (offspring):
Necropsy observations
All pups with scheduled sacrifice on PND 4 were sacrificed by means of CO2 under Isoflurane anesthesia. All pups were examined externally and eviscerated; their organs were assessed macroscopically.
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated further on a caseby-case basis, depending on the type of finding.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
only effect in both sexes: salivation after treatment
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see details
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see details
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Reproductive performance:
no effects observed
Mortality
One female animal of test group 3 (1000 mg/kg bw/d) was sacrificed because it was unable to deliver on GD 24 and showed clinical signs indicative of dystocia. Gross pathology examinations revealed effusion of clear fluid in abdominal and thoracic cavity. All organs examined were without pathological findings. No association of the delivery problem to the treatment is assumed.

Clinical observations for males and females
One high-dose male showed abdominal position after treatment on one day during week 4. One high-dose female showed apathy after treatment on three consecutive days in the first week of administration. These findings are not considered to be a sign of systemic toxicity.
All males of the high- and mid-dose groups and 9 males of the low-dose group showed salivation after treatment (weeks 0 - 5). All females of the high- and mid-dose groups (weeks 0 - 3 and 5 - 7) and 8 females of the low-dose group (weeks 0, 1 and 7) showed salivation after treatment. This salivation was observed for a few minutes immediately after each treatment.

Clinical observations for females during gestation of F1 litters
One high-dose female was sacrificed because it was unable to deliver on GD 24. This rat showed hypothermia, blood crust in anal and/or
genital region, dystocia and undelivered pups palpable in dam’s abdomen. These observations are not considered to be associated to the test compound.
All high- and mid-dose females (GD 0 - 25) and six low-dose females (GD 0, 1, 8, 9, 12 – 14 and 17 – 20) showed salivation after treatment. This salivation was observed for a few minutes immediately after each treatment.
One sperm positive high-dose female, two sperm positive low-dose females and one control female did not deliver F1 pups.

Clinical observations for females and offspring during lactation of F1 litters
All high- and mid-dose dose females (PND 0-4) and one low-dose female (PND 0) showed salivation after treatment during lactation. This salivation was observed for a few minutes immediately after each treatment.
One high-dose dam and one mid-dose dam lost all pups, because they showed insufficient maternal care of pups and did not properly nurse their pups (pups had no or less milk in the stomach).

Food consumption
Food consumption of the high-dose males (1000 mg/kg bw/d) was statistically significantly below control during premating weeks 0 - 1 (about -10%).
Food consumption of the high-dose females was statistically significantly below control during premating weeks 0 - 1 (about 8%) and postnatal days 1 - 4 (about 34%). It was comparable to the concurrent controls during the gestation period.
Mid- and low-dose males and females (100 and 300 mg/kg bw/d) did not show any test substance-related changes of food consumption during the whole treatment period.

Body weight data
The body weights of the high-dose males (1000 mg/kg bw/d) were statistically significantly lower between weeks 2 - 5 (up to 7%) and the body weight change was statistically significantly decreased between weeks 0 - 1 and 4 - 5 (up to 51%). Therefore, body weight change of high-dose males was decreased for whole premating period (about 28% below control). These effects are considered to be treatment-related.
A body weight loss was noted for the high-dose females (-6.2 g) during PND 0 - 4. Body weights and body weight change were comparable to the concurrent controls during premating and gestation period.
Mean body weights and body weight change of the males and females in the low- and middose groups (100 and 300 mg/kg bw/d) were comparable to the concurrent control group throughout the entire study.

Male reproduction data
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls.
Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
One high-dose male, two low dose males and one control male did not generate F1 pups. No histomorphological correlate was found to explain these
apparent infertilities.
Thus, the male fertility index ranged between 80% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

Female reproduction and delivery data
The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until sperm was detected (GD 0) varied between 2.5 and 3.7 days without any relation to exposure.
All sperm positive rats delivered pups or had implants in utero with the following exception: One control female, two low-dose females and one high-dose female did not become pregnant.
The fertility index varied between 80% in low-dose group, 90% in control and high-dose group and 100% in mid-dose group. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
The non pregnant control, low- and high-dose females (0, 100 and 1000 mg/kg bw/d) did not show a histomorphological correlate to explain these apparent infertilities.
The mean duration of gestation was similar in all test groups (i.e. between 22.0 and 22.3 days).
The gestation index was 89% in the high-dose group and 100% in the control, low- and mid-dose group.
Neither the mean number of implantation sites nor the postimplantation loss showed any statistically significant differences between the groups. The postimplantation loss in the highdose group was slightly above the upper limit of historical control data of the test facility (10.9% vs. 9.7% HCD); this is however considered as a by chance finding.
The number of liveborn pups was statistically significantly decreased for high-dose females (71 vs. 98 in control, p ≤ 0.01), resulting from a lower number of pups delivered total (80 vs. 99 in control) and a higher number of stillborn pups (9 vs. 1 in control, p ≤ 0.01). There were also
7 stillborn pups in the mid-dose group (5.6%), this number did not differ statistically significantly from control but slightly above the historical control data (0.0 – 4.5%) Thus live birth indices vary between 89% in high-dose group, 94% in mid-dose group and 99% low-dose and control group.
The rates of liveborn pups and number of stillborn pups in the low-dose females were comparable to control.

Weight parameters
The slight terminal body weight decrease in the male dose group 3 (about -9%) is considered a treatment-related effect. Further significant weight deviations were not calculated.

Gross lesions
The single gross lesions noted are considered to be incidental or spontaneous in nature and not related to treatment.
Two male offspring of test group 2 were suspected to show a dilation of the ductus arteriosus.

Histopathology
All findings noted were either single observations or they were biologically equally distributed between control and treatment groups. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment.
The two offspring suspected for dilation of the ductus arteriosus exhibited an aneurysm of the ductus arteriosus after examination by light
microscopy. This lesion is considered incidental and not related to treatment.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: fertility unaffected
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
high dose: live birth index 89%; mid dose: live birth index 94%
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
high dose: decreased by 18%
Sexual maturation:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see details
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
see details
Pup number and status at delivery
The number of liveborn pups was statistically significantly decreased in high-dose females, resulting from a lower number of pups delivered total and a higher number of stillborn pups. The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were not affected by test substance in mid- and low-dose females.

Pup viability/mortality
The viability index indicating pup mortality during early lactation (PND 0 - 4) was distinctly reduced (-25%, p ≤ 0.01) in the high-dose group (1000 mg/kg bw/d), resulting from significantly higher numbers of died (7 vs. 0 in control, p ≤ 0.01) and cannibalized pups (11 vs. 0 in control, p ≤ 0.01). In the mid-dose group the viability index was reduced (91%, p ≤ 0.01), resulting from a higher number of died pups (5 vs. 0 in control) and a significantly higher number of cannibalized pups (6 vs. 0 in control, p ≤ 0.01).
In the remaining low-dose group viability index was 97%, without showing any association to treatment.

Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Pup clinical observations
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

Pup body weight data
Mean body weights of the high-dose pups (1000 mg/kg bw/d) were statistically significantly below control on PND 1 and PND 4. The average difference to the control was 14% on PND 1 and 17% on PND 4. Mean pup body weight change was slightly, but not statistically significantly, reduced.
No statistically significant changes on F1 pup body weights and body weight change were observed in the mid- and low-dose group (300 and 100 mg/kg bw/d).

Pup necropsy observations
The incidences of empty stomach were slightly increased to 5% and 10% of the pups in the dose group 300 and 1000 mg/kg bw/d, respectively.
A few F1 pups showed spontaneous findings at gross necropsy, such as post mortem autolysis and hemorrhagic testis.
The two offspring of test group 2 suspected for dilation of the ductus arteriosus exhibited an aneurysm of the ductus arteriosus after examination by light microscopy. This lesion was not dose-dependent and, therefore, not considered to be treatment-related.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: correlation between effects on parental animals and offspring questionable
Reproductive effects observed:
not specified

All mid- and high-dose as well as some low-dose animals of both sex showed transient salivation for a few minutes immediately after each treatment. This was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is neither considered to be a sign of systemic toxicity nor as adverse.

Clinical observations indicated distinct toxicity in the exposed parental animals of the highdose group (1000 mg/kg bw/d) but not in the animals of the mid- and low-dose group.

A reduction of food consumption (up to 10% in males and females during treatment weeks 0-1 as well as females (-34%) during lactation), and decreased body weight in males (up to -5%) had been determined during treatment weeks 2-4. A similar pattern as for clinical observations was noted for body weight and body weight change of the parental animals. A distinct decrease was noted in the high-dose animals of both sex even manifested in different time periods of the study. The body weight change in males was reduced from week 0 to 5 (-28% on average in this time period). In females a significant body weight change was observed during lactation leading to a body weight loss (-3%). Consequently, the body weight was decreased in males during treatment weeks 2-5 (-7%) and in females of week 6 (-9%).

The test compound did not adversely affect fertility of the F0 generation parental animals at all dose levels as there were no changes of male/female mating and fertility indices, time until successful copulation, duration of pregnancy and mean number of implantations.

However, there is an alert for a dose-dependent adverse effect of the test substance on pre-/postnatal development of the F1 offspring at mid and high-dose level (300 and 1000 mg/kg bw/d). At the high dose level, this was indicated by a decreased number of delivered/liveborn and an increased number of stillborn pups, resulting in a distinctly reduced pup survival (– 25%). This reduced live birth index was due to losses in only one animal in the group (all others showed no losses). The significantly reduced postnatal offspring weight/weight gain during the first 4 days after birth are likely related to maternal toxicity and ability to care and nurse for the pups as evidenced by clinical observations, empty stomachs in 10% of pups and significantly reduced feed consumption and body weights during the lactation period. Overall, it may be considered that the pup effects seen are secondary to maternal toxicity which effects pup care and nursing.

At the mid dose level, the same effects were noted, but at a lesser incidence and no significant effect on weight/weight gain was observed. The slightly higher (non-significant) number of stillborns may well be contributed to the greater litter size in this group, which leaves the adverse effects on development of offspring in the mid-dose group to be limited to a slightly reduced pup survival (-9%). At least partially, the reduced pup survival may be secondary to a disturbance of maternal care as it became obvious by empty stomachs in pups which have been observed in 5% of mid-dose and 10% of high-dose offspring. In addition,

findings in the mid dose appear to be limited to one animal. This animal appears to have maternal toxicity issues as evidenced by clinical observations (pups not properly nursed and insufficient maternal care of the pups), significantly reduced feed consumption and empty stomachs in the pups. These effects are similar to those seen at the top dose and it may therefore be concluded that maternal toxicity in this one animal is also responsible for the effects seen. The findings in this one animal appear to be an outlier in this group and excluding this animal from the data would show no significant findings vs. controls.

Regarding pathology, the test substance led to no treatment-related changes in the genital organs of males (testes and epididymides) and females (ovaries). These results confirmed that adverse effects of the test substance on fertility were not observed in this study.

Overview of findings diveded by dose group:

High dose: 1000mg/kg

Findings in parental animals:

  • Significantly reduced mean maternal feed consumption during lactation.
  • Reduced mean maternal body weights during gestation (not significant).
  • Reduced mean maternal body weights during lactation (-6.2g from day 0 to 4).
  • Significantly reduced male terminal body weights and during entire study period. No significant change in female terminal body weights.

Findings in pups:

  • Pups not properly nursed, insufficient maternal care of pups and no more pups alive (1 female during lactation).
  • Reduced live birth index (-10%). This is skewed by one single animal which had 9 stillborn pups. All other litters were delivered alive in this group. The reduced overall live births at the top dose is mostly as a consequence of the lower number of initial implantation sites (89 but not statistically significant), followed by post implantation loss (9 - again within the controls) and then 9 stillbirths due to the one animal only. This gives an overall lower figure of 71 viable births vs. controls but not as a consequence of significant findings in the individual steps. One could argue a trend, but there is no clear dose-response in numbers of losses or still-births across the doses.
  • Decreased viability index (-25%), resulting from significantly higher numbers of died (7 vs. 0 in control) and cannibalized pups (11 vs. 0 in control). This was observed across animals (0 to 6 pup deaths) and does not appear to be parent specific. Likely related to reduced maternal feed consumption and body weights during lactation and inadequate nursing.
  • Decreased pup body weights on PND 1 and 4, average difference to the control -14% on PND 1 and -17% on PND 4.Likely related to reduced maternal feed consumption and body weights during lactation and insufficient nursing.
  • Empty stomachs in 10% of pups. Likely related to maternal toxicity and subsequent insufficient maternal care of the pups/inadequate nursing.

 

ConclusionPup effects were clearly seen at this dose as reduced viability and body weights. These are likely related to maternal toxicity and ability to care and nurse for the pups as evidenced by clinical observations, empty stomachs in 10% of pups and significantly reduced feed consumption and body weights during the lactation period. The reduced live birth index was due to losses in only one animal in the group (all others showed no losses). Overall, it may be considered that the pup effects seen are secondary to maternal toxicity which effects pup care and nursing and therefore may not be considered relevant for classification of reproductive toxicity based only on a screening study. 

 

300mg/kg

  • Pups not properly nursed, insufficient maternal care of pups and no more pups alive (1 female during lactation)
  • Slightly increased number of stillborn pups (5.6% vs. 0.0 – 4.5% in historical control data). This is due to 4 deaths seen with one animal (no. 128) and this observation appears to be limited to this one animal at this dose. Animals 130 and 121 showed 2 and 1 stillborns respectively but single deaths were also seen in low dose group and the control. Animal 130 had two pups with aneurysm ductus atreriosis that is quite a rare finding. Question if stillborns also had this condition. This could lead to mortality at a later time point. One pup of animal 130 also had an empty stomach
  • Reduced viability index (-9%), resulting from significantly higher cannibalized pups (6 vs. 0 in control). 10/11 deaths at this dose during lactation are due to animal no. 128. Therefore, this observation was not generalised and is specific to only one animal at this dose in the study. All other dose groups showed viability the same as low dose and control (1 death was seen in animal 126 vs. one in the control). Need to compare this to historic control data to see if there would be the same rate of spontaneous litter loss of a similar fashion. Same applies also to the high dose responder.
  • Empty stomachs in 5% of pups. Almost exclusively due to animal no. 128 (5 pups in animal 128. 1 pup in animal 130).
  • Animal no. 128 showed significantly reduced feed consumption during lactation (9.8 vs. mean of 27.6)! (possibly related to cannibalization of pups as bw change, although low, was within range for this animal There was no significant impact on body weight of the dam, or on body weight gain.)
  • Animal no. 128 was the only animal on this group to show clinical observations of pups not properly nursed and insufficient maternal care of the pups.
  • Excluding findings in animal no. 128, pup body weights, viability and other parameters were no different to controls at the mid dose.
  • Post implantation loss mean %=8.46 vs control =6.24. Not significant.
  • Pups that died during lactation or were stillborn are not statistically different to controls on a group basis.

 

Conclusion Findings in the mid dose appear to be limited to one animal (no. 128). Animal 128 appears to have maternal toxicity issues as evidenced by clinical observations (pups not properly nursed and insufficient maternal care of the pups), significantly reduced feed consumption and empty stomachs in the pups. These effects are similar to those seen at the top dose and it may therefore be concluded that maternal toxicity in this one animal is also responsible for the effects seen. The findings in animal 128 appear to be an outlier in this group and excluding this animal from the data would show no significant findings vs. controls. Need to confirm this to historic control data for a better statistical argument. Overall, it may be considered that the pup effects seen are predominantly limited to one animal only and appear to be secondary to maternal toxicity which effects pup care and nursing. Therefore the relevance of this finding for classification of reproductive toxicity based only on a screening study would be doubtful.

Conclusions:
Since the observed findings show a questionable correlation between the effects seen on maternal toxicity and effects seen on the offspring, their relevance for classification of reproductive toxicity based only on this screening study is doubtful and more complete studies at tolerated doses are required.
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: guideline study acc. to GLP
Justification for type of information:
ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source substances and target substance have similar physical-chemical properties and (eco)toxicological properties because they are either stereoisomers of the target substance, are hydrolysed to the same substance or their chemical structure differs only by an additional double bond. This prediction is supported by data on the substances themselves.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance, L-Citronellol, is a mono-constituent substance (EC No. 231-415-7, CAS no. 7540-51-4 consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and a hydroxyl group. The substance is optically active, comprising a single, pure enantiomeric laevo form.

The source substance, DL-Citronellol, is a mono-constituent substance (EC No. 203-375-0, CAS no. 106-22-9, consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and a hydroxyl group. The substance is an equimolar mixture of two optical isomers (enantiomers).

The source substance, citronellyl acetate, is a mono-constituent substance (EC No. 205-775-0, CAS no. 150-84-5) consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and an acetate group.

The source substance, geraniol and it’s isomer, consist of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. The only difference between the isomers is the position of the first double bond.

The source substance, geraniol and nerol, is a multi-constituent substance of E/Z isomers (EC No. 906-125-5). The constituents consist of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group.

The source substance, geraniol, is a mono-constituent substance (EC No. 203-377-1, CAS no. 106-24-1), consisting of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. Geraniol is a pure form of the E-isomer.

The source substance, nerol, is a mono-constituent substance (EC No. 203-378-7, CAS no. 106-25-2), consisting of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. Nerol is a pure form of the Z-isomer.
The source and target substances are both of high purity with a low concentration of impurities.

3. ANALOGUE APPROACH JUSTIFICATION
The read across hypothesis is based on structural similarity where the source substances only differ in the enantiomeric ratio or an additional double bond. Another source substance is expected to be hydrolysed to the same structure as the target substance.
In a non-chiral environment the target and source chemical DL-Citronellol will have identical properties, but in the chiral environment of living organisms the enantiomers may possess different carcinogenicity and teratogenicity (in a chiral environment, stereoisomers might experience selective absorption, protein binding, transport, enzyme interactions and metabolism, receptor interactions, and DNA binding). All endpoints read-across from DL-Citronellol are considered to be acceptable for this substance assuming that 50% of the target compound is available in the test material.
The source substance citronellyl acetate is read-across from as part of a weight of evidence approach in the repeated dose toxicity endpoint. As this substance is hydrolysed to Citronellol within 2 hours, this read-across endpoint is acceptable in the weight of evidence approach used.
The source substances geraniol, nerol and the reaction mass of geraniol/nerol differ from the target substance only by an additional double bond at C2. These structures are considered to represent a worst case scenario due to the additional potential reactive feature of the second double bond. The genotoxicity, repeated dose and reproductive toxicity endpoints read-across from these substances are therefore acceptable as a worst case assumption.

4. DATA MATRIX
Please refer to the data matrix included in the read-across justification document attached in Section 13.2.
Reason / purpose:
reference to same study
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
dermal application
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Germany
- Age at study initiation: about 11-12 weeks
- Housing: Makrolon cage type M III
- No. of animals per cage: 1 animal,
- Exceptions: during mating: 1 male/ 1 female per cage; during rearing up to PND4: 1 dam with her litter
- Enrichment: Wooden gnawing blocks (Type NGM E-022)
- Bedding: Type Lignocel PS 14 fibres, dustfree bedding
- Diet: Ground Kliba maintenance diet mouse/rat "GLP"; ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12:12
Route of administration:
dermal
Vehicle:
corn oil
Details on exposure:
- Preparation frequency: The preparations were prepared at intervals for which the stability is guaranteed (7 days).
- Application area: Intact clipped skin of the back (dorsal and dorsolateral areas of the trunk; not less than 10% of the body surface); the first clipping was carried out at least 24 hours before the randomization. The rats were reclipped at least once a week (depending on the hair growth).
- Type of application: Dermal application of the test-substance preparations to the clipped intact dorsal skin by means was carried out with 3-mL syringes (3CC Syringe, supplied by Becton, Dickinson & Co., Franklin Lakes, U.S.A.) and a semiocclusive dressing (4 layers of absorbent gauze) and stretch bandage)). The test-substance preparation was applied to the dorsal skin with the syringe in each case. After removal of the dressing, the application area was washed with lukewarm water.
- Volume to be applied: 4 mL/kg body weight (related to the body weight determined most recently in each case)
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up tp 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Application period:
Males: From day 0 (start of administration period) until sacrifice
Females: From day 0 (start of administration period) until GD 19
Frequency of treatment:
daily for at least 6 hours
Dose / conc.:
450 mg/kg bw/day
Remarks:
Doses / Concentrations:
450 mg/kg bw/day
Basis:
nominal conc.
initial high dose
Dose / conc.:
300 mg/kg bw/day
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
nominal conc.
reduced high dose due to massive skin irritation after 10 days of application
Dose / conc.:
150 mg/kg bw/day
Remarks:
Doses / Concentrations:
150 mg/kg bw/day
Basis:
nominal conc.
mid dose
Dose / conc.:
50 mg/kg bw/day
Remarks:
Doses / Concentrations:
50 mg/kg bw/day
Basis:
nominal conc.
low dose
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

DETAILED CLINICAL OBSERVATIONS: Yes
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented for each animal.
The parturition and lactation behavior of the dams was generally evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver or umbilical cord not cut) were documented on an individual dam basis.
On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.
The day of parturition was considered to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day. Deviations from this procedure were on Saturdays, Sundays and public holidays.

BODY WEIGHT:
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning).
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female parental animals:
• During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
• Females without litter were weighed once a week.
• Females between PND 4 and sacrifice were weighed once a week.

FOOD CONSUMPTION:
Generally, food consumption was determined once a week (in a period of 7 days) for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
• Food consumption of the females, which gave birth to a litter, was determined for PND 1-4.
Food consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.
Litter observations:
Pup number and status at delivery
All pups delivered from the F0 parents were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn pups in each litter. At the same time, the pups were examined for gross-morphological changes. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.

Pup viability/ mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays.
The number and percentage of dead pups on the day of birth (PND 0) and pups dying during the lactation period were determined. However, pups which died accidentally and pups which were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day of birth and on PND 4. Furthermore, the viability index was calculated according to the following formula:
Viability index (%) = (number of live pups on day 4 after birth/number of liveborn pups on the day of birth)x 100

Sex ratio
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle. Normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups finally confirmed at necropsy.
The sex ratio was calculated at PND 0 and 4 according to the following formula:
Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4)x100

Pup clinical observations
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned.

Pup body weight data
The pups were weighed one day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results.
Furthermore the body weights on PND 1 were used for the calculation of "runts" (pups, which weighed less than 25% of the mean weight of the respective control pups). The individual weights were always determined at about the same time of the day (in the morning).
In the relevant summary tables pup body weights (including "runts") and pup body weight gains are listed for males, females as well as males and females together.
Postmortem examinations (parental animals):
Necropsy
All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Animals which have died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after their death and assessed by gross pathology.

Organ weights
Weight assessment was carried out on all animals sacrificed at scheduled dates. The following weights were determined:
1. Anesthetized animals
2. Epididymides
3. Testes
4. Ovaries

Organ/tissue fixation
The following organs or tissues of parental animals were fixed in 4% neutral buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Epididymides (fixed in modified Davidson’s solution)
4. Ovaries (fixed in modified Davidson’s solution)
5. Pituitary gland
6. Prostate gland, seminal vesicles, coagulation glands
7. Skin treated
8. Skin untreated
9. Testes (fixed in modified Davidson’s solution)
10. Uterus, oviducts, vagina
The liver, ovaries, testes and epididymides of animals that died or were sacrificed intercurrently were fixed in 4% buffered formaldehyde solution.

Histopathology
After the organs were fixed, histotechnical processing and examination by light microscopy were performed according to the following table:

Organs Test groups
0 1 2 3
All gross lesions A2 A2 A2 A2
Epididymides A1 A1
Ovaries A1 A1
Skin treated A1 A1 A1 A1
Skin untreated A1 A1
Testes A1 A1

A = hematoxylin and eosin stain
1 = all animals per group
2 = all animals affected per group
Animals that have died or were sacrificed in a moribund state were processed histotechnically and assessed like control animals.
Postmortem examinations (offspring):
Pup necropsy observations
All surviving pups (sacrificed on PND 4 under isoflurane anesthesia with CO2), all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
massive skin irritation at 450 mg/kg bw/d
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Lymphocytic infiltrates were observed in treated skin sections which were distributed in an interface pattern
Reproductive performance:
no effects observed
Clinical observations for males
For males, several dermal findings were noted in test groups 3 (450 and 300 mg/kg bw/d). Starting on study day 10, 5 rats showed focal and multifocal red spots on treated skin on several days. Starting on study day 11, 9 male rats showed focal scales on treated skin on several days. One rat showed focal erosion on treated skin on several days beginning on study day 14. Starting on study day 15, 5 rats showed slight erythema on treated skin onseveral days.
Similar findings but less pronounced were observed in male animals of test group 2 (150 mg/kg bw/d), i.e. focal red spots on treated skin on several days in 5 animals from study day 9 onwards, focal scales on treated skin starting on study day 13 in 2 animals and slight erythema on treated skin in 3 animals starting on study day 15.
No treatment-related findings were observed in male animals of test group 1 (50 mg/kg bw/d).

Clinical observations for females
As observed for the male animals of test group 3 (450 and 300 mg/kg bw/d) different dermal findings on treated skin were noted on several days, i.e. during premating, mating and lactation periods. Starting on study day 3, seven animals showed slight and moderate erythema on several days of the study. In addition, some animals of test group 3 (450 and 300 mg/kg bw/d) showed multifocal, focal and diffuse scales on treated skin starting on study day 5.
Similar findings but less pronounced were observed in female animals of test group 2 (150 mg/kg bw/d), i.e. focal red spots, slight erythema and focal scales on treated skin on several days of the study.
In test group 1 (50 mg/kg bw/d) slight erythema as well as focal and diffuse scales on treated skin were observed in individual animals at different time points.
One female of test group 3 (450 and 300 mg/kg bw/d), which did not deliver pups, showed a vaginal hemorrhage 27 days after mating.
In one female animal of test group 2 (150 mg/kg bw/d) a severe thoracal injury was observed during premating. It was a self inflicted injury which was caused by the animal’s attempt to get rid off the gauze. The injury became more severe by time and the animal had to be sacrificed in a moribund state in study week 1. A red-brown lesion was noted on the thorax of this animal, which correlated to an erosion/ulcer on the skin.

Histopathology
Lymphocytic infiltrates were observed in treated skin sections which were distributed in an interface pattern (10/10 males and 5/10 females at 450 mg/kg bw/d [test group 3] graded minimal to slight, 7/10 males at 150 mg/kg bw/d [test group 2] graded minimal, 2/10 males at 50 mg/kg bw/d [test group 1] graded minimal). All other findings noted were single observations which were considered to be incidental and spontaneous in origin and without any relation to treatment.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive performance and fertility
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Gross pathological findings:
no effects observed
Pup number and status at delivery
The mean number of delivered pups per dam and the rate of liveborn and stillborn pups were evenly distributed among test group 0, 1, 2 and 3 (0, 50, 150 and 450 and 300 mg/kg bw/d). The respective values reflect the normal range of biological variation inherent in the strain used in this study.

Pup viability/mortality
No significant findings for pups that died during lactation were observed. The viability index as indicator for pup mortality between PND 0-4 varied between 99% (test group 1, 50 mg/kg bw/d and test group 3, 450 and 300 mg/kg bw/d) and 100% (test group 0, 0 mg/kg bw/d and test group 2, 150 mg/kg bw/d). No test substance-related changes were obtained.
One male pup delivered by one dam of test group 1 (50 mg/kg bw/d) was sacrificed moribund on PND 0 as it showed malformation of the skull, anophthalmia and cleft lip (see Pup clinical observations).

Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show biologically relevant differences between the control and test groups 1-3.

Pup clinical observations
One dam of test group 1 (50 mg/kg bw/d) gave birth to a male pup with a deformation of its snout. On first sight a cleft lip and anophthalmia were seen. The staining of the pups skull showed that several bones (e.g. basisphenoid, palatine, incisive, nasal incl. cartilage and maxilla) were deformed and/or displaced.
Therefore, F1 pups did not show adverse clinical signs up to scheduled sacrifice on PND 4.

Pup body weight data
The mean body weight of female pups in test group 3 (450 and 300 mg/kg bw/d) was increased 11% on PND 1.
Pup body weight changes in the test substance-treated groups 1-3 (50, 150 and 450 and 300 mg/kg bw/d) were comparable to the concurrent control values.
The numbers of runts were 1 female in the control group and 1 male in test group 3 (450 and 300 mg/kg bw/d) on PND 1. Since a dose-response relationship was not observed a relation to dosing was not assumed.

Pup necropsy observations
During the necropsy of all F1 pups no test substance-related findings were observed.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Reproductive effects observed:
not specified
Conclusions:
The dermal administration of Geraniol Extra revealed only local signs of toxicity in male and female Wistar rats at all dose levels. This finding was related to the irritating potential of the test substance.
Thus, concerning toxicity to reproduction the test substance does not need to be classified neither according to Dir 1999/45/EC nor to Reg (EC) 1272/2008.
Executive summary:

Geraniol Extra was administered via dermal administration to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 (vehicle control; test group 0), 50 (test group 1), 150 (test group 2) and 450 mg/kg bw/d (test group 3) in order to observe the possible effects of the test substance on the integrity and performance of the reproductive system in both sexes. Due to severe dermal findings, the dose level for test group 3 was decreased to 300 mg/kg bw/d from study day 10 onwards.

Regarding clinical examinations, only signs of local dermal toxicity were observed for males and females at all dose levels. No changes in food consumption and body weight data were seen at any dose level.

Fertility indices for male and female animals were not impaired by test-substance administration.

Regarding pathology, there were no treatment-related necropsy or histological findings in ovaries, testes or epididymides associated with dermal administration of the test substance. The local minimal inflammatory reactions in the skin of treated males (test groups 1-3) and females (test group 3 only) were regarded as related to treatment and adverse.

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: guideline study acc. to GLP
Justification for type of information:
ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source substances and target substance have similar physical-chemical properties and (eco)toxicological properties because they are either stereoisomers of the target substance, are hydrolysed to the same substance or their chemical structure differs only by an additional double bond. This prediction is supported by data on the substances themselves.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance, L-Citronellol, is a mono-constituent substance (EC No. 231-415-7, CAS no. 7540-51-4 consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and a hydroxyl group. The substance is optically active, comprising a single, pure enantiomeric laevo form.

The source substance, DL-Citronellol, is a mono-constituent substance (EC No. 203-375-0, CAS no. 106-22-9, consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and a hydroxyl group. The substance is an equimolar mixture of two optical isomers (enantiomers).

The source substance, citronellyl acetate, is a mono-constituent substance (EC No. 205-775-0, CAS no. 150-84-5) consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and an acetate group.

The source substance, geraniol and it’s isomer, consist of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. The only difference between the isomers is the position of the first double bond.

The source substance, geraniol and nerol, is a multi-constituent substance of E/Z isomers (EC No. 906-125-5). The constituents consist of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group.

The source substance, geraniol, is a mono-constituent substance (EC No. 203-377-1, CAS no. 106-24-1), consisting of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. Geraniol is a pure form of the E-isomer.

The source substance, nerol, is a mono-constituent substance (EC No. 203-378-7, CAS no. 106-25-2), consisting of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. Nerol is a pure form of the Z-isomer.
The source and target substances are both of high purity with a low concentration of impurities.

3. ANALOGUE APPROACH JUSTIFICATION
The read across hypothesis is based on structural similarity where the source substances only differ in the enantiomeric ratio or an additional double bond. Another source substance is expected to be hydrolysed to the same structure as the target substance.
In a non-chiral environment the target and source chemical DL-Citronellol will have identical properties, but in the chiral environment of living organisms the enantiomers may possess different carcinogenicity and teratogenicity (in a chiral environment, stereoisomers might experience selective absorption, protein binding, transport, enzyme interactions and metabolism, receptor interactions, and DNA binding). All endpoints read-across from DL-Citronellol are considered to be acceptable for this substance assuming that 50% of the target compound is available in the test material.
The source substance citronellyl acetate is read-across from as part of a weight of evidence approach in the repeated dose toxicity endpoint. As this substance is hydrolysed to Citronellol within 2 hours, this read-across endpoint is acceptable in the weight of evidence approach used.
The source substances geraniol, nerol and the reaction mass of geraniol/nerol differ from the target substance only by an additional double bond at C2. These structures are considered to represent a worst case scenario due to the additional potential reactive feature of the second double bond. The genotoxicity, repeated dose and reproductive toxicity endpoints read-across from these substances are therefore acceptable as a worst case assumption.

4. DATA MATRIX
Please refer to the data matrix included in the read-across justification document attached in Section 13.2.
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Germany
- Age at study initiation: about 9 weeks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:
- Housing: Makrolon cage type M III
- No. of animals per cage: 1 animal,
- Exceptions: during mating: 1 male/ 1 female per cage; during rearing up to PND4: 1 dam with her litter
- Enrichment: Wooden gnawing blocks (Type NGM E-022)
- Bedding: Type Lignocel PS 14 fibres, dustfree bedding
- Diet: Ground Kliba maintenance diet mouse/rat "GLP"; ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12:12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
dose volume: 4 ml/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up tp 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Males:
- 14 days premating
- up to 14 days mating
- sacrifice minimum 28 days after first application

Females:
- 14 days premating
- up to 14 days mating
- gestation about 22 days
- sacrifice minimum 4 days after littering
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
Mortality
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied. The examinations of these animals were carried out according to the methods established at the pathology laboratory.

Clinical observations
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal. For technical reasons, however, the clinical observations recorded during the premating period were printed out on a weekly basis. Individual data of daily observations can be found in the raw data.
The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.
On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.
The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

Food consumption
Generally, food consumption was determined once a week (in a period of 7 days) for male and female parental animals, with the following exceptions:
• Food consumption was not determined during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0, 7, 14 and 20.
• Food consumption of F0 females, which gave birth to a litter was determined on PND 1 and 4.
Food consumption was not determined in females without litter during lactation period.

Body weight data
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice.
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
• Females without litter, waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.
Litter observations:
Pup number and status at delivery
The status (sex, liveborn or stillborn) and number of all delivered pups were determined as soon as possible on the day of birth. At the same time, the pups were also examined for macroscopically evident changes. Pups that die before this initial examination are defined as stillborn pups.

Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods, which are described in detail in section 3.8.2.4. “Necropsy observations”.
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. The viability index was calculated according to the following formula:
Viability index (%) = (number of live pups on day 4 after birth/number of live pups on the day of birth) x100

Sex ratio
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy.
The sex ratio was calculated at day 0 and day 4 after birth according to the following formula:
Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) x100

Pup clinical observations
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned.

Pup body weight data
The pups were weighed on the day after birth (PND 1) and on PND 4.
Pups' body weight change was calculated from these results.
The individual weights were always determined at about the same time of the day (in the morning).
“Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.
Postmortem examinations (parental animals):
Necropsy
All animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Special attention was given to the reproductive organs.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Epididymides
3. Testes
4. Ovaries

Organ / Tissue fixation of parental animals
The following organs or tissues of parental animals were fixed in neutral buffered 4%
formaldehyde or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Pituitary gland
4. Testis (fixed in modified Davidson’s solution)
5. Epididymides (fixed in modified Davidson’s solution)
6. Prostate gland, seminal vesicles, coagulation glands
7. Ovaries (fixed in modified Davidson’s solution)
8. Uterus, oviducts, vagina
The uteri of all cohabited female F0 parental animals were examined for the presence and number of implantation sites.
The uteri of apparently nonpregnant animals or empty uterus horns were placed in 10% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method). Then the uteri were rinsed carefully under running water. When the examinations are completed, the uteri were transferred to the Pathology Laboratory for further processing.

Histopathology of parental animals
After the organs were fixed, histotechnical processing and examination by light microscopy was performed according to the following table:

Organs Test groups
0 1 2 3
1. All gross lesions A2 A2 A2 A2
2. Testes A1 A1
3. Epididymides A1 A1
4. Ovaries A1 A1

Methods and scope of examination:
A = hematoxylin and eosin stain
1 = all animals per group
2 = all animals affected per group

The hematoxylin-eosin stained slides were examined by light microscopy and assessed. A correlation between gross lesions and histopathological findings was performed.
Postmortem examinations (offspring):
Necropsy observations
All pups with scheduled sacrifice on PND 4 were sacrificed by means of CO2 under Isoflurane anesthesia. All pups were examined externally and eviscerated; their organs were assessed macroscopically.
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated further on a caseby-case basis, depending on the type of finding.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
only effect in both sexes: salivation after treatment
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see details
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see details
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Reproductive performance:
no effects observed
Mortality
One female animal of test group 3 (1000 mg/kg bw/d) was sacrificed because it was unable to deliver on GD 24 and showed clinical signs indicative of dystocia. Gross pathology examinations revealed effusion of clear fluid in abdominal and thoracic cavity. All organs examined were without pathological findings. No association of the delivery problem to the treatment is assumed.

Clinical observations for males and females
One high-dose male showed abdominal position after treatment on one day during week 4. One high-dose female showed apathy after treatment on three consecutive days in the first week of administration. These findings are not considered to be a sign of systemic toxicity.
All males of the high- and mid-dose groups and 9 males of the low-dose group showed salivation after treatment (weeks 0 - 5). All females of the high- and mid-dose groups (weeks 0 - 3 and 5 - 7) and 8 females of the low-dose group (weeks 0, 1 and 7) showed salivation after treatment. This salivation was observed for a few minutes immediately after each treatment.

Clinical observations for females during gestation of F1 litters
One high-dose female was sacrificed because it was unable to deliver on GD 24. This rat showed hypothermia, blood crust in anal and/or
genital region, dystocia and undelivered pups palpable in dam’s abdomen. These observations are not considered to be associated to the test compound.
All high- and mid-dose females (GD 0 - 25) and six low-dose females (GD 0, 1, 8, 9, 12 – 14 and 17 – 20) showed salivation after treatment. This salivation was observed for a few minutes immediately after each treatment.
One sperm positive high-dose female, two sperm positive low-dose females and one control female did not deliver F1 pups.

Clinical observations for females and offspring during lactation of F1 litters
All high- and mid-dose dose females (PND 0-4) and one low-dose female (PND 0) showed salivation after treatment during lactation. This salivation was observed for a few minutes immediately after each treatment.
One high-dose dam and one mid-dose dam lost all pups, because they showed insufficient maternal care of pups and did not properly nurse their pups (pups had no or less milk in the stomach).

Food consumption
Food consumption of the high-dose males (1000 mg/kg bw/d) was statistically significantly below control during premating weeks 0 - 1 (about -10%).
Food consumption of the high-dose females was statistically significantly below control during premating weeks 0 - 1 (about 8%) and postnatal days 1 - 4 (about 34%). It was comparable to the concurrent controls during the gestation period.
Mid- and low-dose males and females (100 and 300 mg/kg bw/d) did not show any test substance-related changes of food consumption during the whole treatment period.

Body weight data
The body weights of the high-dose males (1000 mg/kg bw/d) were statistically significantly lower between weeks 2 - 5 (up to 7%) and the body weight change was statistically significantly decreased between weeks 0 - 1 and 4 - 5 (up to 51%). Therefore, body weight change of high-dose males was decreased for whole premating period (about 28% below control). These effects are considered to be treatment-related.
A body weight loss was noted for the high-dose females (-6.2 g) during PND 0 - 4. Body weights and body weight change were comparable to the concurrent controls during premating and gestation period.
Mean body weights and body weight change of the males and females in the low- and middose groups (100 and 300 mg/kg bw/d) were comparable to the concurrent control group throughout the entire study.

Male reproduction data
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls.
Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
One high-dose male, two low dose males and one control male did not generate F1 pups. No histomorphological correlate was found to explain these
apparent infertilities.
Thus, the male fertility index ranged between 80% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

Female reproduction and delivery data
The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until sperm was detected (GD 0) varied between 2.5 and 3.7 days without any relation to exposure.
All sperm positive rats delivered pups or had implants in utero with the following exception: One control female, two low-dose females and one high-dose female did not become pregnant.
The fertility index varied between 80% in low-dose group, 90% in control and high-dose group and 100% in mid-dose group. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
The non pregnant control, low- and high-dose females (0, 100 and 1000 mg/kg bw/d) did not show a histomorphological correlate to explain these apparent infertilities.
The mean duration of gestation was similar in all test groups (i.e. between 22.0 and 22.3 days).
The gestation index was 89% in the high-dose group and 100% in the control, low- and mid-dose group.
Neither the mean number of implantation sites nor the postimplantation loss showed any statistically significant differences between the groups. The postimplantation loss in the highdose group was slightly above the upper limit of historical control data of the test facility (10.9% vs. 9.7% HCD); this is however considered as a by chance finding.
The number of liveborn pups was statistically significantly decreased for high-dose females (71 vs. 98 in control, p ≤ 0.01), resulting from a lower number of pups delivered total (80 vs. 99 in control) and a higher number of stillborn pups (9 vs. 1 in control, p ≤ 0.01). There were also
7 stillborn pups in the mid-dose group (5.6%), this number did not differ statistically significantly from control but slightly above the historical control data (0.0 – 4.5%) Thus live birth indices vary between 89% in high-dose group, 94% in mid-dose group and 99% low-dose and control group.
The rates of liveborn pups and number of stillborn pups in the low-dose females were comparable to control.

Weight parameters
The slight terminal body weight decrease in the male dose group 3 (about -9%) is considered a treatment-related effect. Further significant weight deviations were not calculated.

Gross lesions
The single gross lesions noted are considered to be incidental or spontaneous in nature and not related to treatment.
Two male offspring of test group 2 were suspected to show a dilation of the ductus arteriosus.

Histopathology
All findings noted were either single observations or they were biologically equally distributed between control and treatment groups. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment.
The two offspring suspected for dilation of the ductus arteriosus exhibited an aneurysm of the ductus arteriosus after examination by light
microscopy. This lesion is considered incidental and not related to treatment.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: fertility unaffected
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
high dose: live birth index 89%; mid dose: live birth index 94%
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
high dose: decreased by 18%
Sexual maturation:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see details
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
see details
Pup number and status at delivery
The number of liveborn pups was statistically significantly decreased in high-dose females, resulting from a lower number of pups delivered total and a higher number of stillborn pups. The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were not affected by test substance in mid- and low-dose females.

Pup viability/mortality
The viability index indicating pup mortality during early lactation (PND 0 - 4) was distinctly reduced (-25%, p ≤ 0.01) in the high-dose group (1000 mg/kg bw/d), resulting from significantly higher numbers of died (7 vs. 0 in control, p ≤ 0.01) and cannibalized pups (11 vs. 0 in control, p ≤ 0.01). In the mid-dose group the viability index was reduced (91%, p ≤ 0.01), resulting from a higher number of died pups (5 vs. 0 in control) and a significantly higher number of cannibalized pups (6 vs. 0 in control, p ≤ 0.01).
In the remaining low-dose group viability index was 97%, without showing any association to treatment.

Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Pup clinical observations
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

Pup body weight data
Mean body weights of the high-dose pups (1000 mg/kg bw/d) were statistically significantly below control on PND 1 and PND 4. The average difference to the control was 14% on PND 1 and 17% on PND 4. Mean pup body weight change was slightly, but not statistically significantly, reduced.
No statistically significant changes on F1 pup body weights and body weight change were observed in the mid- and low-dose group (300 and 100 mg/kg bw/d).

Pup necropsy observations
The incidences of empty stomach were slightly increased to 5% and 10% of the pups in the dose group 300 and 1000 mg/kg bw/d, respectively.
A few F1 pups showed spontaneous findings at gross necropsy, such as post mortem autolysis and hemorrhagic testis.
The two offspring of test group 2 suspected for dilation of the ductus arteriosus exhibited an aneurysm of the ductus arteriosus after examination by
light microscopy. This lesion was not dose-dependent and, therefore, not considered to be treatment-related.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: correlation between effects on parental animals and offspring questionable
Reproductive effects observed:
not specified

All mid- and high-dose as well as some low-dose animals of both sex showed transient salivation for a few minutes immediately after each treatment. This was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is neither considered to be a sign of systemic toxicity nor as adverse.

Clinical observations indicated distinct toxicity in the exposed parental animals of the highdose group (1000 mg/kg bw/d) but not in the animals of the mid- and low-dose group.

A reduction of food consumption (up to 10% in males and females during treatment weeks 0-1 as well as females (-34%) during lactation), and decreased body weight in males (up to -5%) had been determined during treatment weeks 2-4. A similar pattern as for clinical observations was noted for body weight and body weight change of the parental animals. A distinct decrease was noted in the high-dose animals of both sex even manifested in different time periods of the study. The body weight change in males was reduced from week 0 to 5 (-28% on average in this time period). In females a significant body weight change was observed during lactation leading to a body weight loss (-3%). Consequently, the body weight was decreased in males during treatment weeks 2-5 (-7%) and in females of week 6 (-9%).

The test compound did not adversely affect fertility of the F0 generation parental animals at all dose levels as there were no changes of male/female mating and fertility indices, time until successful copulation, duration of pregnancy and mean number of implantations.

However, there is an alert for a dose-dependent adverse effect of the test substance on pre-/postnatal development of the F1 offspring at mid and high-dose level (300 and 1000 mg/kg bw/d). At the high dose level, this was indicated by a decreased number of delivered/liveborn and an increased number of stillborn pups, resulting in a distinctly reduced pup survival (– 25%). This reduced live birth index was due to losses in only one animal in the group (all others showed no losses). The significantly reduced postnatal offspring weight/weight gain during the first 4 days after birth are likely related to maternal toxicity and ability to care and nurse for the pups as evidenced by clinical observations, empty stomachs in 10% of pups and significantly reduced feed consumption and body weights during the lactation period. Overall, it may be considered that the pup effects seen are secondary to maternal toxicity which effects pup care and nursing.

At the mid dose level, the same effects were noted, but at a lesser incidence and no significant effect on weight/weight gain was observed. The slightly higher (non-significant) number of stillborns may well be contributed to the greater litter size in this group, which leaves the adverse effects on development of offspring in the mid-dose group to be limited to a slightly reduced pup survival (-9%). At least partially, the reduced pup survival may be secondary to a disturbance of maternal care as it became obvious by empty stomachs in pups which have been observed in 5% of mid-dose and 10% of high-dose offspring. In addition,

findings in the mid dose appear to be limited to one animal. This animal appears to have maternal toxicity issues as evidenced by clinical observations (pups not properly nursed and insufficient maternal care of the pups), significantly reduced feed consumption and empty stomachs in the pups. These effects are similar to those seen at the top dose and it may therefore be concluded that maternal toxicity in this one animal is also responsible for the effects seen. The findings in this one animal appear to be an outlier in this group and excluding this animal from the data would show no significant findings vs. controls.

Regarding pathology, the test substance led to no treatment-related changes in the genital organs of males (testes and epididymides) and females (ovaries). These results confirmed that adverse effects of the test substance on fertility were not observed in this study.

Overview of findings diveded by dose group:

High dose: 1000mg/kg

Findings in parental animals:

  • Significantly reduced mean maternal feed consumption during lactation.
  • Reduced mean maternal body weights during gestation (not significant).
  • Reduced mean maternal body weights during lactation (-6.2g from day 0 to 4).
  • Significantly reduced male terminal body weights and during entire study period. No significant change in female terminal body weights.

Findings in pups:

  • Pups not properly nursed, insufficient maternal care of pups and no more pups alive (1 female during lactation).
  • Reduced live birth index (-10%). This is skewed by one single animal which had 9 stillborn pups. All other litters were delivered alive in this group. The reduced overall live births at the top dose is mostly as a consequence of the lower number of initial implantation sites (89 but not statistically significant), followed by post implantation loss (9 - again within the controls) and then 9 stillbirths due to the one animal only. This gives an overall lower figure of 71 viable births vs. controls but not as a consequence of significant findings in the individual steps. One could argue a trend, but there is no clear dose-response in numbers of losses or still-births across the doses.
  • Decreased viability index (-25%), resulting from significantly higher numbers of died (7 vs. 0 in control) and cannibalized pups (11 vs. 0 in control). This was observed across animals (0 to 6 pup deaths) and does not appear to be parent specific. Likely related to reduced maternal feed consumption and body weights during lactation and inadequate nursing.
  • Decreased pup body weights on PND 1 and 4, average difference to the control -14% on PND 1 and -17% on PND 4.Likely related to reduced maternal feed consumption and body weights during lactation and insufficient nursing.
  • Empty stomachs in 10% of pups. Likely related to maternal toxicity and subsequent insufficient maternal care of the pups/inadequate nursing.

 

ConclusionPup effects were clearly seen at this dose as reduced viability and body weights. These are likely related to maternal toxicity and ability to care and nurse for the pups as evidenced by clinical observations, empty stomachs in 10% of pups and significantly reduced feed consumption and body weights during the lactation period. The reduced live birth index was due to losses in only one animal in the group (all others showed no losses). Overall, it may be considered that the pup effects seen are secondary to maternal toxicity which effects pup care and nursing and therefore may not be considered relevant for classification of reproductive toxicity based only on a screening study. 

 

300mg/kg

  • Pups not properly nursed, insufficient maternal care of pups and no more pups alive (1 female during lactation)
  • Slightly increased number of stillborn pups (5.6% vs. 0.0 – 4.5% in historical control data). This is due to 4 deaths seen with one animal (no. 128) and this observation appears to be limited to this one animal at this dose. Animals 130 and 121 showed 2 and 1 stillborns respectively but single deaths were also seen in low dose group and the control. Animal 130 had two pups with aneurysm ductus atreriosis that is quite a rare finding. Question if stillborns also had this condition. This could lead to mortality at a later time point. One pup of animal 130 also had an empty stomach
  • Reduced viability index (-9%), resulting from significantly higher cannibalized pups (6 vs. 0 in control). 10/11 deaths at this dose during lactation are due to animal no. 128. Therefore, this observation was not generalised and is specific to only one animal at this dose in the study. All other dose groups showed viability the same as low dose and control (1 death was seen in animal 126 vs. one in the control). Need to compare this to historic control data to see if there would be the same rate of spontaneous litter loss of a similar fashion. Same applies also to the high dose responder.
  • Empty stomachs in 5% of pups. Almost exclusively due to animal no. 128 (5 pups in animal 128. 1 pup in animal 130).
  • Animal no. 128 showed significantly reduced feed consumption during lactation (9.8 vs. mean of 27.6)! (possibly related to cannibalization of pups as bw change, although low, was within range for this animal There was no significant impact on body weight of the dam, or on body weight gain.)
  • Animal no. 128 was the only animal on this group to show clinical observations of pups not properly nursed and insufficient maternal care of the pups.
  • Excluding findings in animal no. 128, pup body weights, viability and other parameters were no different to controls at the mid dose.
  • Post implantation loss mean %=8.46 vs control =6.24. Not significant.
  • Pups that died during lactation or were stillborn are not statistically different to controls on a group basis.

 

Conclusion Findings in the mid dose appear to be limited to one animal (no. 128). Animal 128 appears to have maternal toxicity issues as evidenced by clinical observations (pups not properly nursed and insufficient maternal care of the pups), significantly reduced feed consumption and empty stomachs in the pups. These effects are similar to those seen at the top dose and it may therefore be concluded that maternal toxicity in this one animal is also responsible for the effects seen. The findings in animal 128 appear to be an outlier in this group and excluding this animal from the data would show no significant findings vs. controls. Need to confirm this to historic control data for a better statistical argument. Overall, it may be considered that the pup effects seen are predominantly limited to one animal only and appear to be secondary to maternal toxicity which effects pup care and nursing. Therefore the relevance of this finding for classification of reproductive toxicity based only on a screening study would be doubtful.

Conclusions:
Since the observed findings show a questionable correlation between the effects seen on maternal toxicity and effects seen on the offspring, their relevance for classification of reproductive toxicity based only on this screening study is doubtful and more complete studies at tolerated doses are required.
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
Quality of whole database:
Read across from reaction mass of Nerol (cis-3,7-dimethyl-2,6-octadien-1-ol ) and Geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol).
Effect on fertility: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Read across from Geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol).

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Read across from reaction mass of Nerol (cis-3,7-dimethyl-2,6-octadien-1-ol ) and Geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol).
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Read across from Geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol).

Justification for classification or non-classification

 

Reproductive toxicity includes adverse effects on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring.

A dermal reproductive toxicity study was conducted according to OECD guideline 421 at concentrations 50, 150, 300 and 450 mg/kg bw/day of Geraniol. Due to severe dermal findings, the dose level for test group 3 was decreased to 300 mg/kg bw/d from study day 10 onwards. No treatment-related effects were observed for the reproductive performance or litter response paramaters examined. Therefore a 'No Observed adverse Effect Level' (NOAEL) for reproductive toxicity was considered to be 300 mg/kg bw/day.

An oral reproductive toxicity study was conducted according to OECD guideline 421 at concentrations 100, 300 and 1000 mg/kg bw/day Nerol/Geraniol. No treatment related effects were observed for reproductive performance, however, the live birth index and viability index of the pups was decreased at 300 and 1000 mg/kg bw/day. At least partially, the reduced pup survival in the mid dose group may be secondary to a disturbance of maternal care as it became obvious by empty stomachs in pups which have been observed in 5% of mid-dose and 10% of high-dose offspring. In addition, findings in the mid dose appear to be limited to one animal. This animal appears to have maternal toxicity issues as evidenced by clinical observations (pups not properly nursed and insufficient maternal care of the pups), significantly reduced feed consumption and empty stomachs in the pups. These effects are similar to those seen at the top dose and it may therefore be concluded that maternal toxicity in this one animal is also responsible for the effects seen.

Based on the results of these studies there is no evidence that the substance has an adverse effect on sexual function and fertility. There is evidence of an effect on development at the mid and high doses in the oral study. Findings in the mid dose appear to be limited to one animal (no. 128). Animal 128 appears to have maternal toxicity issues as evidenced by clinical observations (pups not properly nursed and insufficient maternal care of the pups), significantly reduced feed consumption and empty stomachs in the pups. These effects are similar to those seen at the top dose and it may therefore be concluded that maternal toxicity in this one animal is also responsible for the effects seen. The findings in animal 128 appear to be an outlier in this group and excluding this animal from the data would show no significant findings vs. controls. Overall, it may be considered that the pup effects seen are predominantly limited to one animal only and appear to be secondary to maternal toxicity which effects pup care and nursing. Therefore the relevance of this finding for classification of reproductive toxicity based only on a screening study would be doubtful. The substance is therefore not classified for reproductive toxicity according to the available data.