3,7-dimethylocta-2,6-dienyl acetate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information is used for read-across to Neryl acetate multi.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (2E)-3,7-dimethylocta-2,6-dien-1-ol and (2Z)-3,7-dimethylocta-2,6-dien-1-ol

EC Number:

906-125-5

Molecular formula:

Unspecified

IUPAC Name:

Reaction mass of (2E)-3,7-dimethylocta-2,6-dien-1-ol and (2Z)-3,7-dimethylocta-2,6-dien-1-ol

Details on test material:

- Name of test material (as cited in study report): Geraniol 60
- Physical state: liquid, colorless, clear
- Stability under test conditions: guaranteed by the sponsor
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Details on results (P0)

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.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: fertility unaffected

Remarks on result:

other: Key result for fertility

2

Key result

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:

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

Details on results (F1)

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.

Effect levels (F1)

Key result

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

Remarks on result:

other: In the presence of the OECD TG 414 study the NOAEL for developmental toxicity will not be used for further assessment

Overall reproductive toxicity

Key result

Reproductive effects observed:

not specified

Any other information on results incl. tables

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.

 

Conclusion –Pup 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.

Applicant's summary and conclusion

Conclusions:

The NOAEL for reproduction (fertility) is >=1000 mg/kg bw/d in the presence of maternal toxicity, with a NOAEL of 300
mg/kg bw.
The developmental toxicity seen in this study at 100 mg/kg w, is not used for the asessment but the results of the OECD TG 414 are used for this.

Executive summary:

For Geraniol / Nerol (60/40% reaction mass 60; Geraniol 60) data is available from a study according to OECD TG 421 and in compliance with GLP criteria. In this study, rats were exposed by gavage to 100, 300 or 1000 mg/kg bw/day of the test item in corn oil. Based on significantly lower body weight (gain) of high animals, the parental NOAEL was concluded to be 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. Hence, the respective NOAEL for fertility is >=1000 mg/kg bw/day for this Geraniol /Nerol (Geraniol 60), whereas the NOAEL for systemic effects is set at 300 mg/kg bw.

Developmental toxicity in this Reproscreen study: The number of live born 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. Furthermore, the viability index indicating pup mortality during early lactation (PND 0 - 4) was reduced in the high and mid dose group. Fetal weights were reduced in the high dose group. Based on these effects the NOAEL for development was considered to be 100 mg/kg bw/day, based on decreased pup viability in the group exposed to 300 mg/kg bw/day in absence of maternal toxicity.

Body weight effects were seen on pups and therefore the developmental toxicity in this study at 100 mg/kg bw, which were further investigated in an OECD TG 414 study.