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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study acc. to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
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)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
liquid, colorless, clear

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
450 mg/kg bw/day
Basis:
nominal conc.
initial high dose
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
nominal conc.
reduced high dose due to massive skin irritation after 10 days of application
Remarks:
Doses / Concentrations:
150 mg/kg bw/day
Basis:
nominal conc.
mid dose
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

Examinations

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.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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 function / performance (P0)

Reproductive performance:
no effects observed

Details on results (P0)

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.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
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)
Sex:
male/female
Basis for effect level:
other: systemic toxicity

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: developmental toxicity

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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.