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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:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Golpanol PAP / 2-Propyn-1-ol, compd. with methyloxirane
- Physical state: liquid
- Analytical purity: 54.7 area %
- Impurities (identity and concentrations): 33.9 g/100 g water, propargyl alcohol 0.6 area %, propylene glycol 5.3 area %, higher alcoxylated products 5.4 area %
- Lot/batch No.: 88767924U0
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor,
and the sponsor holds this responsibility.
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Horst, The Netherlands
- Age at study initiation: At the start of the treatment period the animals were 11-12 weeks of age
- Weight at study initiation: the initial body weight variation did not exceed ±20% of the mean weight for each sex
- Housing: During the
premating period, the animals were housed in groups of 4/sex. For mating, one male and one female were housed together. Mated females were housed individually in macrolon cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days prior to the experimental start date

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2oC
- Humidity (%): 45 -65 %
- Air changes (per hr): about 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered to the animals daily by oral gavage. A dosing volume of 2 mL/kg body weight was used for all groups. Dosing volume was adjusted based on the latest body weight. For the females during gestation the dosing volume remained constant from gestation day 14 up to the end of gestation. The vehicle (drinking water) was used for dosing the control group and was used for diluting the test item to the appropriate concentrations.
Dilutions of the test substance in the vehicle and the vehicle for dosing the controls were prepared weekly (namely 26 April, 2, 9, 16, 23 and 30 May, 6 and 13 June 2012), and stored in a refrigerator (2-10ºC), in portions sufficient for one day.
Male animals were dosed during a 2-week premating period, and during mating and up to the day of sacrifice. The female animals were dosed during a 2-week premating period, and during mating, gestation and lactation up to the day of sacrifice (approx. day 4 of lactation).

VEHICLE
- Amount of vehicle (if gavage): dosing volume was 2 mL/kg bw/day
Details on mating procedure:
At the end of the premating period, each female was caged with one male from the same group. Animals were caged together until mating occured or 1 week had elapsed.
Mating pairs were clearly identified. Every consecutive morning during the mating period, vaginal smears were made for determination of the presence of sperm. The day on which sperm was detected in the vaginal smear was considered as gestation day 0. Upon evidence of copulation the females were caged individually for the birth and rearing of their pups.
Sperm positive females that turn out to be non-pregnant were killed not earlier than 21 days after copulation. Females that did not show evidence of copulation after the end of the oneweek mating period were be housed individually until sacrifice (not earlier than 21 days after the last day of the mating period). Dams were allowed to raise their litter until sacrifice on day 4 of lactation or shortly thereafter.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity was determined in the dosign formulation from 30 may 2012 and content was determined in the dosing formulations from 2 and 30 May 2012 according to a validated method of analysis.
Duration of treatment / exposure:
Male animals were dosed during a 2-week premating period, and during mating and up to the day of sacrifice. The female animals were dosed during a 2-week premating period, and during mating, gestation and lactation up to the day of sacrifice (approx. day 4 of lactation).
Frequency of treatment:
once daily
Details on study schedule:
N/A
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
of the test material as supplied by the sponsor
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Remarks:
of the test material as supplied by the sponsor
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
of the test material as supplied by the sponsor
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a 14-day range-finding study
- Rationale for animal assignment (if not random): the animals (males and females separately) were allocated to the various groups by computer randomization proportionately to body weight.
Positive control:
N/A

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS / DETAILED CLINICAL OBSERVATIONS:
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. All cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. All abnormalities, signs of ill health or reactions to treatment were recorded according, but not limited to the listing of clinical signs in annex 4 to the study plan. Any animal showing signs of
severe debility or intoxication, particularly if death appears imminent, would be humanely killed to prevent loss of tissues by cannibalism or autolytic degeneration.

BODY WEIGHT: Yes
Body weights of male and female animals were recorded shortly before the start of administration of the test item at randomization and at the start of the study (day 0). Males were weighed weekly until sacrifice.
Females were weighed once per week during the premating period. Mated females were weighed on days 0, 7, 14 and 21 during presumed gestation and on day 0 and 4 of lactation.
Non-mated females were weighed once per week after the mating period.
In addition, the animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION:
Feed consumption was measured per cage over weekly intervals during the study, with exeptance of the mating period, during which no feed consumption was registered. The results were expressed in g per animal per day and g per kg body weight per day.
Oestrous cyclicity (parental animals):
N/A
Sperm parameters (parental animals):
N/A
Litter observations:
To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.
The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups was evaluated on days 0 and 4 of lactation. The pups were individually weighed on days 0 and 4 of lactation. Mean pup weight was calculated per sex and for both sexes combined.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Male animals were dosed during a 2-week premating period, and during mating and up to the day of sacrifice.
- Maternal animals: Dams were allowed to raise their litter until sacrifice on day 4 of lactation or shortly thereafter.

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY / ORGAN WEIGHTS: Yes

All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. Male animals were sacrificed after the mating period. Female animals were sacrificed at or shortly after day 4 of lactation. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which were preserved in Bouin's fixative:
- ovaries (after counting the corpora lutea)
- uterus (after counting of the implantation sites)
- testes
- epididymides,
- seminal vesicles
- prostate
- all gross lesions

In addition of 5 animals/sex/group taken from 3 cages for the males and taking into account the pregnancy status of the females, the following organs were preserved:
adrenals [w]
bone marrow (femur)
brain [w] (including sections of cerebrum, cerebellum, medulla/pons)
caecum
cervix
clitorial gland
colon
coagulation gland
duodenum
eyes
femur including joint
heart [w]
ileum
jejunum (including Peyer's patches)
kidneys [w]
liver [w]
lungs
mesenterial and axillary lymph nodes
ovaries [w]
peripheral nerve (sciatic or tibial)
pituitary gland
preputial gland
prostate [w]
rectum
seminal vesicles [w](including coagulation gland)
skeletal muscle (thigh)
spinal cord (cervical, mid-thoracic and lumbar)
spleen [w]
stomach*
thymus [w]
thyroid [w] (including parathyroid)
trachea
urinary bladder
uterus [w]
vagina
* Non glandular (“forestomach”) and glandular (fundus, pyorus) parts were examined microscopically.
[w] These organs were weighed (paired organs together) as soon as possible after dissection to avoid drying.
Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 um, and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS haematoxylin. Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high concentration group (group 4). If treatment-related changes were observed in the high-dose group, the evaluation of these tissues/organs was extended to the intermediate-dose groups (2 and 3).
In addition, reproductive organs of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and middose groups, were microscopically examined.
Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.
Postmortem examinations (offspring):
Grossly malformed pups were sacrificed and examined. A necropsy was performed on stillborn pups and pups dying during the study; macroscopic abnormalities were recorded. At necropsy of the dams, at or shortly after day 4 of lactation, pups were examined externally for gross abnormalities and killed by appropriate techniques.
Statistics:
The resulting data were analyzed using the methods mentioned below. Other statistical tests may be performed when considered appropriate. Tests were generally performed as twosided tests with results taken as significant where the probability of the results is p<0.05 (*) or p<0.01 (**).
- Continuous data were subjected to a decision tree or parametric statistical test.
- Dichotomous data were evaluated using the statistical test Chi-square-Fisher.
Statistical profiles are presented in amendment 2 in Annex 7 to this report.
Reproductive indices:
see any other information on materials and methods
Offspring viability indices:
see any other information on materials and methods

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no unscheduled deaths. No treatment related clinical signs were observed in males during the premating, mating and postmating period or in females during the premating, mating, gestation and lactation period.
The tail showed a missing tip or encrustations in several animals at random incidence. Upon healing of the wound, the tail was complete and no further observations were recorded.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No effects on body weight or body weight change were observed in males and females throughout the study.
In the first week of the premating phase, mean food consumption was slightly, but statistically significantly lower in the males of the high dose group, both expressed per animal or per kg body weight. In the second week of the premating phase, the food intake per kg body weight was increased in this group. These slight fluctuations in food intake in the high dose males at the beginning of a study are considered chance findings. No significant effects on food consumption were observed in males post-mating of in females throughout the study.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
In each dosing group 12 females were placed with 12 males, resulting in 10/12 mated females in the mid dose group and 12/12 mated females in the other dosing groups, respectively.
One mated female in the high dose group (1/12) did not deliver, but showed implantation sites at autopsy and was therefore considered pregnant.
The mean number of corpora lutea and the mean number of implantation sites was comparable in all groups. The mean pre-implantation loss and the mean prenatal loss was comparable in all groups.
All females survived delivery, resulting in 12/12, 12/12, 10/12 and 11/12 litters in the control group, low dose, mid dose and high dose groups, respectively. All females delivered litters with liveborn pups. Three litters (3/12) in the control group and 2 litters (2/10) in the mid dose group comprised stillborn pups. There were no litters comprising stillborn pups only.

ORGAN WEIGHTS (PARENTAL ANIMALS)
A statistically significantly increased mean absolute and relative liver weight was observed in the males and the females of the high dose group.In addition, relative (but not absolute) liver weight was statistically significantly increased in the males of the mid dose group.
Mean absolute and relative kidney weight was statistically significantly increased in the males and the females of the high dose group.
An increase in mean absolute and relative prostate weight was statistically significant in the males of the mid dose and high dose group. In view of the rather low prostate weight values in the control group and in absence of histopathological changes on the prostate, this was considered to be a chance finding.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At necropsy no treatment related gross changes were observed.
One female in the mid dose group (#49) showed macroscopic changes in the liver and marked focal hepatocellular necrosis. In addition, this female showed high AST and ALT values, indicating hepatocellular damage. However, in absence of histopathological findings in the liver at the high dose group, the liver effects of female 49 are considered to be spontaneous.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Microscopic examinations of the sampled organs and tissues did not reveal treatment related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only.
They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
parental, development and reproduction
Effect level:
125 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: non adverse effects on liver and kidney

Results: F1 generation

Details on results (F1)

Litter data
Litter size and sex
The mean number of pups delivered per litter was comparable in all groups (11.1, 11.9, 12.7 and 13.0 in the control, low dose, mid dose and high dose groups, respectively). The mean percentage male littermates at delivery was comparable in all groups (51.5, 57.3, 46.4, 50.3 in the control, low dose, mid dose and high dose group, respectively).

Pup observations
One male pup in the control group and one male pup in the low dose group showed a wound. Seven males and 9 female pups were cold on lactation day 0, all these pups belonging to the same litter (female 37) in the low dose group. These findings in one litter in the low dose group are not ascribed to treatment.

Pup body weight
Mean pup body weight was comparable for males and females in all groups on day 0 of lactation. On day 4 of lactation, mean pup body weight was statistically significantly lower in the females of the mid dose group. However, in absence of a dose response relationship, this is not considered treatment-related.

Viability index
A total number of 130, 143, 125 and 143 pups were liveborn in the control, low dose, mid dose and high dose group, respectively. Three pups in the control group and 2 pups in the mid dose group were stillborn and a total of 18, 13, 5 and 6 pups in the control, low dose, mid dose and high dose group, respectively, were missing or had died on lactation day 4, resulting in viability indices on lactation day 4 of 86%, 93%, 94% and 96% for the control, low dose, mid dose and high dose group, respectively. One female (number 21) in the control group showed complete litter loss.

Macroscopic observations of stillborn pups or pups that died during lactation
Macroscopic examination of stillborn pups showed no milk in the stomach and/or no distention of the lungs in two pups in the mid dose group and no milk in the stomach in three pups in the control group.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed in the F1 animals at this dose level

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

The objective of this study was to provide initial data on the possible effects of the test item 2 - propyn-1-ol with methyloxirane on reproductive performance of Wistar rats and the development of pups consequent to daily oral administration of the test item via gavage at concentrations of 0, 5, 25 or 125 mg/kg to male and female rats during a premating period of 2 weeks and during mating (1 week), up to a total of approximately 4 weeks for males and including gestation and lactation until postnatal day 4 (PN day 4) for females (up to a total of approximately 6 weeks).

The 2-propyn-1-ol was homogeneously distributed in the gavage liquids. Results from content analysis showed slightly higher, but acceptable concentrations of the gavage liquids as compared to the nominal concentration.

Daily clinical observations during the premating, gestation and lactation period or neurobehavioural observations and motor activity assessment at the end of the study did not reveal any treatment-related changes in the animal’s appearance, general condition or

behaviour.

No treatment-related effects were observed in mean body weight, body weight changes and food consumption in 2-propyn-1-ol compound with methyl oxirane-exposed animals throughout the study.

No treatment-related effects were observed on mating index, male and female fertility indices, gestation index, duration of gestation, number of corpora lutea, implantation sites, lost implantations. No effects were observed on litter size, pup sex and weight and pup survival.

In males a marked treatment-related increase of kidney weight and liver weight were observed at the 125 mg/kg bw group, which was accompanied by a slight increase in alanine aminotransferase activity, bilirubin levels and bile acids. However, in absence of histopathological changes in the liver and kidney, these effects are considered treatment-related, but not adverse.

In females a marked treatement-related increase of kidney weight and liver weight were observed at the 125 mg/kg bw group. However, in absence of histopathological changes in the liver and kidney, these effects are considered treatment-related, but not adverse.

For males the No Observed Adverse Effect Level (NOAEL) for systemic toxicity is established at the high dose level of 125 mg/kg bw 2-propyn-1-ol compound with methyl oxirane, based on the absence of adverse effects. Based on the treatment-related related effects observed on kidney weight and liver weight and parameters, the No Observed Effect Level (NOEL) for males is established at the low dose of 5 mg/kg bw.

For males the NOAEL for fertility is established at the highest dose of 125 mg/kg bw, because no effects were seen on male fertility.

For females the NOAEL for systemic toxicity is established at the high dose of 125 mg/kg bw, based on the absendce of adverse effects.

Based on treatment-related effects on liver weight and kidney weight the No Observed Effect Level (NOEL) for females is established at the mid dose of 25 mg/kg bw.

For females the NOAEL for fertility and development is established at the highest dose of 125 mg/kg bw), because no effects were seen on female fertility and development.

Applicant's summary and conclusion