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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-04-11 to 2014-07-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und dLebensmittelsicherheit, München, Germany)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Test System
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: approx. 12-13 weeks old
Body weight at the allocation of the animals to the experimental groups:interval within ± 20% of the mean weight, if technically possible.
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act
on Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3°C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0902)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological
controls at regular intervals)
- The animals were kept individually in IVC cages (except during the mating period when two females were paired with one male), type III H,
polysulphone cages on Altromin saw fibre bedding (lot no. 230113)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days)

Number and Sex of the Animals
156 animals (52 males and 104 females) were included in the study.

Preparation of the Animals
After the acclimatisation period of at least 5 days, females were paired with males as per the ratio of 1:2 (male to female). Prior to the start of the
mating a detailed clinical observation outside the home cage was made. None of the animal showed pathological signs before the initiation of
mating period.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Details on exposure:
The test item was weighed into a tarred plastic vial on a precision balance and the required volume of sesame oil was added and further
vortexing it for 2-3 minutes.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration.
The test item formulation was prepared freshly on each administration day before the administration procedure.
The vehicle was also used as control item.

The following doses were evaluated:
Control: 0 mg/kg body weight
Low Dose: 100 mg/kg body weight
Medium Dose: 300 mg/kg body weight
High Dose: 1000 mg/kg body weight

The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence
of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL. The doses were selected on the basis of data
from a Dose Range Finding Study (BSL study no. 130404).

The animals in the control group were handled in an identical manner to the test group subjects and received sesame oil using the same
volume as used for the high dose group.

The test item formulation or vehicle was administered at a single dose to the animals by oral gavage.
The application volume for all groups was 4 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The assessment of homogeneity as well as a determination of the nominal concentration of the test item in the vehicle were performed at
various intervals.
Samples for analysis of the dose formulations of the test item in the vehicle (nominal concentration) were taken in the first and last week of the
study for all doses.
Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation. Samples were taken in the first and last week of the study.
All formulation samples were stored at -20° C and were analysed after completion of the in-life phase of the toxicity study at BSL BIOSERVICE
Scientific Laboratories GmbH.
Details on mating procedure:
Females were paired for cohabitation in batches in order to regularise the number of animals for terminal sacrifice on a particular day.
The subsequent morning and the next morning onwards, the vaginal smear of female was checked to confirm the pregnancy.
The day on which sperms were observed in the vaginal smear was considered as gestation day ‘0’. Mated females were assigned in an unbiased
manner to the control and treatment groups ensuring that group mean body weights are comparable with each other.
Each animal was assigned a unique identification number. 52 males and 104 females were included in the study instead. Out of 104 females,
95 were mated and distributed 24 each in control, MD, HD and 23 in LD group. Non mated females were discarded without any further observations.
Duration of treatment / exposure:
The test item was orally administered daily in graduated doses to several groups of pregnant females from the gestation day (GD) 5
to gestation day (GD) 19
Frequency of treatment:
Once per day. 7 days per week
Duration of test:
On GD 20, i.e. the day prior to the expected day of delivery, the presumed pregnant females are subjected to a caesarean section.
No. of animals per sex per dose:
156 animals (52 males and 104 females) were included in the study. The 4 groups comprised 24 each in control, MD, HD and 23 in LD group. Five non mated females were discarded without any further observations.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL. Selection was based on a preliminary dose-range-finding study.
- Rationale for animal assignment (if not random): Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that the mean body weights were comparable to each other

Examinations

Maternal examinations:
Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly
during the treatment.
The sperm positive females were weighed during gestations days 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except
on the day of their arrival.
Food consumption of pregnant females was measured on gestations days 5, 8, 11, 14, 17 and 20.
Food consumption was measured neither for males during the entire study nor for both male and females during the mating period.

Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of
anticipated effects after dosing. The health condition of the animals was recorded. At least once daily all animals were observed for morbidity
and mortality.
Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Post-Mortem Examination
On gestation day 20, sperm positive were subjected to a caesarean section after sacrificing the animals using an overdose of pentobarbital
injected intraperitoneally (Narcoren®, Merial; lot no.: 228112; expiry date: 30/11/2015).
At the time of termination, the dam (presumably pregnant female) was examined macroscopically for any structural abnormalities or
pathological changes which may have influenced the pregnancy.
Immediately after the termination or as soon as possible after death, the uteri were removed and the pregnancy status of the dams was confirmed.
Uteri that appear non-gravid were further examined by staining with 10 % ammonium sulphide solution to confirm the non-pregnant status.
Each gravid uterus with the cervix was weighed.
The number of corpora lutea was counted for pregnant animals. The uterine contents were examined for embryonic or foetal deaths as well as
the number of viable foetuses. The degree of resorption (late and early) was confirmed in order to help estimate the relative time of death
of the conceptus. The position and number of foetuses in each uterine horn was also recorded.
Males were sacrificed without any observations any time after the mating.

Foetal Evaluations
All foetuses from a particular dam were identified by using different colour strings and were weighed and sexed based on the anogenital distance.
Each foetus was examined for external anomalies.
One half of each litter was processed by Alizarin red staining and examined for skeletal alterations. The remaining litter was examined for soft tissue anomalies by a microdissection technique.
Craniofacial examination of the heads of the foetuses used for the soft tissue examination was performed for internal structure including the eyes,
brain, nasal passage and tongue by razor blade serial sectioning technique.
For interpretation of significant external, visceral, and skeletal findings, they were described as developmental variations (alterations in anatomic
structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence,
representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or
interfere with normal body function, or may be incompatible with life).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
A statistical assessment of the results of the body weight, food consumption was performed by comparing values of dosed with control animals
using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters
were analysed using a Chi-square test. The statistics are performed with GraphPad Prism V.6.01 software or IDBS Workbook 8.1.2, ANOVA v8.8
software (p<0.05 is considered as statistically significant).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
All sperm positive females survived until the scheduled necropsy.
Minor clinical signs like moving the bedding , piloerection , salivation were observed on few isolated days thoughout the dose groups. Additionally, few spontaneous clinical signs, including alopecia , eschar and slight nasal discharge were observed in all dose groups including control animals. These various clinical signs in the control and treatment groups were not considered to be adverse.
None of the female showed signs of abortion or premature delivery prior to the scheduled sacrifice.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Prenatal Data
The statistical analysis of prenatal data revealed no significant effect on prenatal parameters like gravid uterus weight, number of corpora lutea,
live foetuses, dead foetuses, resorptions (early and late), sex ratio, percent preimplantation loss, post implantation loss, group mean number of male and female foetuses in the treated groups when compared with controls. However, a statistically significant decrease in group mean terminal
body weight and adjusted maternal weight was observed in HD group when compared to the control. This effect on terminal body weight and adjusted maternal weight in HD group could be attributed to treatment with test item.

Litter Data
No statistically significant and treatment-related effects were observed on group mean litter weight, total litter weight, male litter weight,
female litter weight, group mean number of live foetuses, number of males and number of females were observed when compared to the
control group.

Foetal Evaluation
No gross external abnormalities were seen in control and treatment groups.
Skeletal examination of the Alizarin red stained foetuses revealed a range of findings which were of a type or which occurred at an incidence
comparable/ lower in treated groups when compared to the control group. As these skeletal findings were minor variations and due to the lack of dose dependency and consistency in these findings indicates no treatment-related adverse effects. There was no indication of a test item-related trend in the type and incidences of other findings and they were therefore, considered to be spontaneous in nature.
Internal examinations of the foetal viscera by the free-hand-microdissection technique revealed a range of visceral abnormalities in all groups
including the control. All observed findings are considered minor variations, and due to lack of dose dependency no significant toxicological significance can b attributed to these findings and they are considered to be spontaneous in nature.
Craniofacial examination by a razor blade serial sectioning technique revealed a range of visceral findings in all groups including controls. These findings are not considered as treatment-related and solely spontaneous in nature.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In this prenatal developmental toxicity study, the repeated dose administration of 1-Hexanol, 2 ethyl-, manuf. of, by products from, distn Residues
to pregnant female Wistar rats at doses of 100, 300 and 1000 mg/kg body weight from gestation day 5 to 19 revealed no major toxicological findings in terms of mortality but few clinical signs in MD and HD group and statistically significant effects on female body weight and food consumption was observed in HD group.
In foetuses, no test item related effect on foetal parameters was observed in treatment groups when compared with the controls.
There were few skeletal findings mainly related to ossification observed and evenly distributed between all dose groups including controls.
These skeletal findings are considered to be minor variations which are not associated with long term consequences on survival, general growth and development. Due to the lack of dose dependency and consistency, these skeletal findings were not considered to be treatment-related.

Based on the findings from this study, The NOAEL of 1-Hexanol, 2 ethyl-, manuf. of, by products from, distn Residues in Wistar rat for
maternal toxicity is considered to be 300 mg/kg body weight/day and for foetal toxicity as 1000 mg/kg body weight/day.
Executive summary:

For the registration substance a guideline conform OECD 414 prenatal developmental toxicity study was performed following the principles of GLP. The aim of this study was to assess possible adverse effects on pregnant females and embryo-foetal development which could arise from repeated exposure of 1-Hexanol, 2 ethyl-, manuf. of, by products from, distn Residues, via oral administration (gavage) to female rats during the gestation days 5 to 19. Nulliparous and non-pregnant females were mated with males (2:1 ratio) and divided into four groups based on their body weights on the day of sperm positive vaginal smears (GD 0). The 4 groups comprised 24 animals each in control, MD, HD and 23 in LD group. Animals of the control group were handled identically as the dose groups, but received sesame oil, the vehicle used in this study.

Analysis of formulation samples for nominal concentration and homogeneity revealed stable and homogenious formulations over the period they were used

During the period of administration and also during pre-exposure gestation days (0-4), the animals were observed precisely each day for signs of toxicity and mortality. All female animals were sacrificed on the respective gestation day 20. Following the gross necropsy, the uteri and ovaries were removed, weighed and examined for number of implantations, resorptions (early and late) live and dead foetuses. Foetuses were identified by color strings, sexed and weighed. All foetuses were observed for external abnormalities, half of the Foetuses for visceral and craniofacial abnormalities and the remaining half of the litter was observed for skeletal abnormalities. The sperm positive females were weighed during gestations days 0, 5, 8, 11, 14, 17 and 20. Food consumption of sperm positive females was measured on gestations days 5, 8, 11, 14, 17 and 20. The uteri of the non-pregnant females were processed with 10 % ammonium sulphide solution and checked for the early embryonic deaths.

The following doses were evaluated:

Control:                        0         mg/kg bw/day

Low Dose:                    100     mg/kg bw/day

Medium Dose:              300    mg/kgbw/day

High Dose:                   1000  mg/kgbw/day

The test item formulation was prepared freshly on each day of administration. The test item was suspended in sesame oil and administered daily during the gestation days 5 and 19 to female animals.

Summary Results

All sperm positive females survived until the scheduled necropsy.

Minor test item-related clinical signs like moving the bedding, piloerection and salivation were observed on few , isolated days in all dose groups. There were also few sponstaneous clinical signs like alopecia, eschar and slight nasal discharge observed. All of these findings are considered to be not adverse. None of the female showed signs of abortion or premature delivery prior to the scheduled sacrifice.

Statistical analysis of body weight and body weight gain data during gestation period revealed statistically significant decrease in body weight on gestation day 11, 17 and 20 in HD group when compared with the controls. Overall body weight gain during 0-20 in HD group was also decreased (although statistical significance was not achieved) when compared with controls. In correlation to the body weight and body weight gain, the food consumption was comparable in the C, LD and MD groups. However, in HD group, statistically significant decrease in food consumption was observed from GD 5 throughout the study period when compared with controls.

The statistical analysis of prenatal data revealed no significant effect on prenatal parameters like gravid uterus weight, number of corpora lutea, live foetuses, dead foetuses, resorptions (early and late), sex ratio, percent preimplantation loss, post implantation loss, group mean number of male and female foetuses in the treated groups when compared with controls.

No statistically significant and treatment-related effects were observed on group mean litter weight, total litter weight, male litter weight, female litter weight, group mean number of live foetuses, number of males and number of females were observed when compared to the control group.

No gross external abnormalities were seen in control and treatment groups.

Skeletal examination of the Alizarin red stained foetuses revealed a range of findings which were of a type or which occurred at an incidence comparable/ lower in treated groups when compared to the control group. However, all of these skeletal findings were minor variations which are not associated with long term consequences on survival, general growth and developmentand. Due to the lack of any dose dependency, these skeletal findings were not considered to be treatment-related. Internal examinations of the foetal viscera by the free-hand-microdissection technique revealed a range of visceral abnormalities in all groups including the control. .


Conclusion

In this prenatal developmentaltoxicity study,the repeated dose administration of 1-Hexanol, 2 ethyl-, manuf. of, by products from, distn Residues to pregnant female Wistar rats at doses of 100, 300 and 1000 mg/kg body weight from gestation day 5 to 19 revealed no major toxicological findings in terms of mortality but few minor clinical signs in MD and HD group not considered to be adverse. Statistically significant effects on female body weight and food consumption were observed in animals from the HD group. 

In foetuses, no test item related effect on foetal parameters was observed in treatment groups when compared with the controls. There were few skeletal findings mainly related to ossification observed and evenly distributed between all dose groups including controls. These skeletal findings are considered to be minor variations which are not associated with long term consequences onsurvival, general growth and development. Due to the lack of dose dependency and consistency, these skeletal findings were not considered to be treatment-related.

Based on the findings from this study, the NOAEL of 1-Hexanol, 2 ethyl-, manuf. of, by products from, distn Residues in Wistar rat for maternal toxicity is considered to be 300 mg/kg body weight/day and for foetal toxicity as 1000 mg/kg body weight/day.