Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: dermal
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: acc. to OECD guideline and GLP
Cross-reference
Reason / purpose:
reference to same study

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:
other: OECD Guideline 421 (Reproduction/Developmental Toxicity Screening test)
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

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

Type of coverage:
semiocclusive
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)
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Mating details:
- M/F ratio per cage: 1:1
- Length of cohabitation: up tp 14 days

Application period:
Males: From day 0 (start of administration period) until sacrifice
Females: From day 0 (start of administration period) until GD 19
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Frequency of treatment:
daily for at least 6 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
450 mg/kg bw/d
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
300 mg/kg bw/d
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
150 mg/kg bw/d
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
50 mg/kg bw/d
Basis:
nominal per unit body weight
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
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.
Sacrifice and pathology:
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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Dermal irritation:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
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

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: The only effect seen was skin irritation.

Target system / organ toxicity

Critical 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.