Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-12-28 to 2012-02-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is regarded as reliable without restrictions because it was conducted in compliance with GLP regulation and guideline.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
22 March 1996
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test as of July 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
NOTOX B.V. Hambakenwetering 7 5231 DD's-Hertogenbosch The Netherlands
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 11 weeks
- Housing: in Macrolon plastic cages (MIV type, height 18 cm)
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water
- Acclimation period: At least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2 – 22.2°C
- Humidity (%): 25 - 74%
- Air changes: 15 air changes per hour
- Photoperiod: 12 hours artificial light and 12 hours darkness per day
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity/density of the test substance. No correction was made for the purity of the test substance.

Method: Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: 14 days
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted during the treatment phase according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

The formulations of Group 2 and Group 4 were homogeneous (coefficient of variation < 10%) and stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

On 05-01-12, the Group 2, Group 3 and Group 4 formulations used for dosing on 04-01-12 were analysed. The concentrations analysed were below target (i.e. 78% - 89%).

On 26-01-12, the Group 2, Group 3 and Group 4 formulations used for dosing were analysed. The concentrations analysed in the formulation of Group 2 and Group 4 were in agreement with target concentrations (i.e. mean accuracy between 90% and 110%). The concentrations analyzed in the formulation of Group 3 were below target (i.e. 58%).

On 06-02-12, the Group 2, Group 3 and Group 4 formulations used for dosing were analysed. The concentrations analysed in the formulation of Group 2 were in agreement with target concentrations (i.e. mean accuracy between 90% and 110%). The concentrations analyzed in the formulation of Group 3 and Group 4 were below target (i.e. 68% and 82%).

On 22-02-12, the Group 2, Group 3 and Group 4 formulations were prepared for analysis only. The concentrations analysed in the formulation of Group 2 were in agreement with target concentrations (i.e. mean accuracy between 90% and 110%). The concentrations analyzed in the formulation of Group 3 and Group 4 were below target (i.e. 75% and 88%).

All formulations were prepared correctly and no analytical or other practical reason could be given for the variability in the analyses or the low accuracies obtained. Concentrations over a greater range were determined to be accurate in the 14-Day pilot study NOTOX Project 498336; BASF Project 01R0658/01X035). As such, the results were ultimately accepted and the results were attributed to the nature of the test substance itself. Furthermore, the formulations were consistently accurate at the 5 mg/kg dose level, which was the determined NOAEL. There was no impact on the study’s integrity
Duration of treatment / exposure:
Males were exposed for 29-31 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females were exposed for 38-43 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
Remarks:
Doses / Concentrations:
0, 5, 20, 60 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on results from a 14-day dose range finding study where 0, 10, 25, 60 and 120 mg/kg bw/day were administered. Several haematology and clinical biochemistry parameters were affected at 60 and/or 120 mg/kg bw/day, and higher liver and spleen weights (absolute and relative) were noted at these dose levels as well. Enlarged spleens were noted for all animals at 120 mg/kg bw/day, and foci on the stomach and thickened limiting ridge of the stomach were also commonly noted at this dose level. Collectively, the data at 120 mg/kg is suggestive of haemolytic anaemia. Based on these results, 5, 20 and 60 mg/kg bw/day were chosen for the main study.
Positive control:
not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made for all animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. These clinical observations were at least conducted between 2.5 and 4 hours after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, clinical observations, body weight, gross necropsy

GROSS EXAMINATION OF DEAD PUPS:
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
Postmortem examinations (parental animals):
Pathology
All males and the selected 5 females/group were deprived of food overnight (with a maximum of 20 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.

Necropsy was conducted on the following days:
- Females which delivered: Lactation Days 5-7
- Females which failed to deliver: Post-coitum Day 30
- Males: Following completion of the mating period (a minimum of 28 days of dose administration)
- Spontaneous deaths: As soon as possible after death and always within 24 hours
- Euthanized in extremis: When pain, distress or discomfort is considered not transient in nature or is likely to become more severe

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. The numbers of former implantation sites and corpora lutea were recorded for all paired females. Samples of the following tissues and organs were collected from all animals and fixed in 10% buffered formalin: Ovaries, Adrenal glands, Adrenal glands, Peyer's patches, Brain - cerebellum, mid-brain, cortex, Pituitary gland,Caecum,Preputial gland, Cervix, Prostate gland, Clitoral gland, Rectum, Colon, Salivary glands - mandibular, sublingual, Coagulation gland, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides, Skeletal muscle, Eyes, Spinal cord -cervical, midthoracic, lumbar, Spleen, Femur including joint, Sternum with bone marrow, Heart, Stomach (forestomach and glandular stomach), Ileum,Testes 2, Jejunum, Thymus,Kidneys, Thyroid including parathyroid if detectable, Lacrimal gland, exorbital, Tongue, Larynx, Trachea, Liver, Urinary bladder, Lung, infused with formalin, Uterus,Lymph nodes - mandibular, mesenteric, Vagina, All gross lesions
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

Organ weights
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
- Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix),Kidneys , Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, Thyroid including parathyroid

Histopathology
All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.
Of all the males of the control and high dose group and all males suspected to be infertile or which died before mating, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes was processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin.
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4
- The additional slides of the testes of all males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis
- The preserved organs and tissues of the animals of all dose groups which died spontaneously or were killed in extremis.
- All gross lesions of all animals (all dose groups)
- Stomach, spleen and sternum of all animals of Groups 2 and 3 (males and females), based on (possible) treatment-related changes in these organs in Group 4
- Liver and kidneys of all male animals of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
- The reproductive organs of all animals of Groups 1 and 4 and from male no. 22 and female no. 62 (20 mg/kg bw/day) who did not have live offspring.




Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at 4 days of age.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding.

Necropsy pups
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

The following additional methods of statistical analysis were used:
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores). Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
- Male mating index
- Male fertility index
- Female mating index
- Female fertility index
- Gestation index
Offspring viability indices:
- Live birth index
- Postimplantation loss
CLINICAL SIGNS AND MORTALITY
One female was killed in extremis and one female was found dead at 60 mg/kg bw/day. Both of these animals were noted with necrosis of the glandular stomach at the microscopic examination, which was possibly related to treatment.
No clinical signs of toxicity were noted during the observation period. Piloerection was noted for a single high dose female on one day during the study. At the incidence observed, it was not considered to be toxicologically relevant.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically relevant changes in body weights and body weight gain were noted.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
Males at 60 mg/kg bw/day had significantly lower red blood cells (RBC), haemoglobin, and mean corpuscular haemoglobin concentration (MCHC), along with increased reticulocytes and mean corpuscular volume (MCV). Females at this dose level had significantly increased neutrophils (also seen for females at 20 mg/kg bw/day, but was not statistically significant) and decreased lymphocytes (also seen for females at 20 mg/kg bw/day, but did not reach statistical significance). These data collectively suggest a mild regenerative macrocytic hypochromic anemia.

There were no other toxicologically relevant changes in haematology parameters.

The statistically significant increase in prothrombin time (PT) seen for females at 20 mg/kg bw/day was attributable to slightly low control values and did not reflect a treatment related effect.

CLINICAL CHEMISTRY
Males at 60 mg/kg bw/day had significantly lower alkaline phosphatase (ALP) and significantly higher inorganic phosphate levels than controls. Clinical biochemistry parameters were unaffected for females at this dose level. The changes for males were not considered to be biologically relevant.

There were no other treatment related effects on clinical biochemistry parameters.

The significant increase in glucose seen for males at 20 mg/kg bw/day was not considered to be toxicologically relevant as it occurred in the absence of a dose related distribution

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
Total movements were significantly lower for females at 60 mg/kg bw/day. Total activity was lower than controls during intervals 6-8, though the difference from controls was only slight, there were no significant effects on ambulatory activity, and in the absence of any relevant clinical observations like lethargy or reduced activity, the difference between controls was not considered to be toxicologically relevant.

ORGAN WEIGHTS
Absolute and relative liver and spleen weights were higher for animals for both sexes at 60 mg/kg bw/day (absolute liver weights were not significantly higher for females at this level). At 20 mg/kg bw/day, absolute liver weights were higher for animals of both sexes, though the difference was not significantly different from controls. Liver to body weight ratios were significantly higher for males at this dose level, however

GROSS PATHOLOGY
At 60 mg/kg bw/day one female was found dead and at the macroscopic examination was noted with the GI tract distended with gas, beginning autolysis, gelatinous contents of the GI tract, many greenish foci on the stomach, enlarged adrenal glands, two fetuses and two resorptions in the right uterine horn, and seven fetuses in the left uterine horn.
Enlarged lungs, pale discoloration of the lungs and watery clear fluid in the thoracic cavity were noted for one female that was euthanized in extremis. These are indicative of a gavage error. This female was also noted with four living fetuses in the left uterine horn and eight living fetuses in the right uterine horn.

Macroscopic findings noted for animals at 60 mg/kg bw/day that survived to the end of the scheduled treatment period included black discoloration of the spleen, noted for five males and two females. Additionally, dark red foci on the stomach glandular mucosa, enlarged spleen and/or liver were noted for a few animals at this dose level. Reduced size and black brown discoloration of the liver papillary process was seen for one male.
There were no treatment related effects on the macroscopic examination up to 20 mg/kg bw/day


HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related microscopic findings were present in:
- Stomach: hyperplasia of the limiting ridge was present at an increased incidence and severity in male and female rats treated at 20 (Group 3; 5/5 and 3/5 rats respectively) and 60 (Group 4; 8/8 and 5/6 rats respectively) mg/kg bw/day up to a moderate degree compared to minimal degrees in control (Group 1; 2/5 male rats) and 5 mg/kg bw/day (Group 2; 2/5 and 2/5 rats respectively) treated rats
- Stomach: hyperkeratosis of the limiting ridge was present in male and female rats treated at 20 (2/8 and 2/5 rats respectively) and 60 (2/5 and 5/6 rats respectively) mg/kg bw/day.
- Spleen: hemopoietic foci, primarily erythropoiesis were present at an increased severity in male and female rats treated at 60 mg/kg bw/day (7/7 and 8/8 respectively) up to a marked degree. The severity was higher compared to gradings noted up to a moderate degree in control (5/5 rats both sexes), in 5 (5/5 rats both sexes) and in 20 (5/5 rats both sexes) mg/kg bw/day treated rats. This was the microscopic correlate to the macroscopically enlarged spleens.
- Spleen: hemosiderin pigment was present at an increased incidence and severity in male rats treated at 60 mg/kg bw/day (7/7) up to a marked degree compared to a minimal degree noted for control (3/5), 5 mg/kg bw/day (4/5) and 20 mg/kg bw/day (4/5) treated rats. There was an increased mean severity seen for female rats treated at 60 mg/kg bw/day (8/8) of slight to moderate degree compared to a minimal to slight degree in control (5/5), in 5 mg/kg bw/day (5/5) and 20 mg/kg bw/day (5/5) treated rats. This was the microscopic correlate to the macroscopic black discolouration.
- Sternal bone marrow: erythroid hyperplasia was present in male and female rats treated at 60 mg/kg bw/day (5/5 and 5/6 respectively) up to a slight degree
- Liver: centrilobular hepatocellular hypertrophy, was present at a slight degree in the 60 mg/kg bw/day treated female that was found dead. In males, this was present at an increased incidence and severity in 20 (4/5) and 60 (5/5) mg/kg bw/day treated rats up to a slight degree, compared to minimal degrees in control (1/5) and 5 mg/kg bw/day (2/5) treated male rats.
- Kidneys: hyaline droplets were present at an increased incidence and severity in males treated at 60 mg/kg bw/day (5/5) up to a moderate degree, compared to minimal to slight degrees present in control (3/5), 5 (3/5) and 20 (5/5) mg/kg bw/day treated male rats.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No toxicologically relevant effects on the gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: - changes in haematology parameters, macroscopic findings and organ weights, - adverse microscopic alterations in the stomach, spleen and liver - microscopic findings in the kidneys and bone marrow
Remarks on result:
other: Generation: P general systemic toxicity (migrated information)
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Remarks on result:
other: Generation: P: reproductive performance and fertility (migrated information)
VIABILITY (OFFSPRING)
The number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.
The number of dead pups at first litter check represented 0 and 2.2% for controls and animals at 60 mg/kg bw/day, respectively while the postnatal loss represented 3.1 and 0% of living pups for controls and high dose animals, respectively.
Two pups of the control group went missing and one pup of the control group and two pups at 60 mg/kg bw/day were found dead during the first days of lactation. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS (OFFSPRING)
Small size, an incidental finding, was the only clinical sign noted. This was not toxicologically relevant.

BODY WEIGHT (OFFSPRING)
Body weights of pups were unaffected by treatment up to 60 mg/kg bw/day.

GROSS PATHOLOGY (OFFSPRING)
The only incidental macroscopic findings for pups that were found dead was the absence of milk in the stomach. Wound on the left front leg and shoulder were the only findings noted at macroscopic examination for surviving pups. These were incidental in nature and no toxicological relevance was attributed to any of these findings
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and guideline compliant study.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Reproduction toxicity via oral route

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental toxicity screening test according to OECD TG 422 was to provide initial information concerning the toxic potential of 3-Hexyne-2,5-diol and on its possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with oral administration to rats at repeated doses. 3-Hexyne-2,5-diol was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 5, 20 and 60 mg/kg/day (n=10/sex/group). Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29-31days). The females were exposed for 2 weeks prior to mating, during mating, duringpost-coitum, and at least 4 days of lactation (for 38-43 days). Homogeneity and stability of formulations were demonstrated by analyses. The accuracy results were below target for all treatment groups during the first analysis. Subsequent analyses were conducted yielding acceptable results at 5 mg/kg bw/day, and variable results at 20 and 60 mg/kg bw/day. No reason could be determined for the variable results or low accuracies obtained. The concentration at which the NOAEL was determined was verified by analysis. As such, the results were ultimately accepted and the results were attributed to the nature of the test substance itself.

Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of pups. Five dams and males cohabited with were selected from each group for further toxicity examinations such as functional observations, haematology, clinical chemistry, gross necropsy, organ weighing and histopathology. The dams were allowed to litter, and rear their young up to termination on days 4 – 7 postpartum. Pups were weighed and observed for possible abnormalities. All parental animals were subjected to gross pathology one day after the last treatment and offspring were euthanized. Selected organs were weighed. Full histopathology was performed in 5 animals/sex/group. The reproductive organs and pituitary of non-pregnant female animals and males cohabited with in the low and mid dose groups were also processed and evaluated histologically. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (water) only.

Results

Parental toxicity was seen for animals of both sexes at 20 and 60 mg/kg bw/day. Toxicity was characterized by changes in haematology parameters, macroscopic findings and organ weights for animals at 60 mg/kg bw/day and by adverse microscopic alterations in the stomach, spleen and liver at 20 and 60 mg/kg bw/day. Animals of the high dose also had microscopic findings in the kidneys and bone marrow. Taken together, the test substance caused hemolysis and a regenerative response of the bone marrow. The liver and spleen changes were related to hemolysis, and mild anemia was evident at 60 mg/kg bw/day.

No toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, and clinical biochemistry). No reproduction toxicity was observed up to the highest dose level tested (60 mg/kg bw/day).No developmental toxicity was observed up to the highest dose level tested (60 mg/kg bw/day).

Conclusion

In conclusion, treatment with 3-Hexyne-2,5-diol by oral gavage in male and female Wistar Han rats at dose levels of 5, 20 and 60 mg/kg body weight/day revealed parental toxicity at 20 and 60 mg/kg body weight/day. No reproduction and developmental toxicity was observed for treatment up to 60 mg/kg body weight/day. Based on these results, the following No Observed Adverse Effect Levels (NOAEL) was derived: 5 mg/kg bw bw/day. The NOAEL for reproductive parameters were determined to be 60 mg/kg bw/day.


Short description of key information:
Treatment with 3-Hexyne-2,5-diol by oral gavage in male and female Wistar Han rats at dose levels of 5, 20 and 60 mg/kg body weight/day revealed parental toxicity at 20 and 60 mg/kg body weight/day. No reproduction toxicity was observed for treatment up to 60 mg/kg body weight/day. Based on these results, the following No Observed Adverse Effect Levels (NOAEL) was derived: 5 mg/kg bw bw/day. The NOAEL for reproductive parameters were determined to be 60 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
Only one Study available

Effects on developmental toxicity

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb 2016 - May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 440/2008
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: B102 06.11.2015
- Purity: 98.8 corr. area%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 10-12 weeks
- Weight at study initiation: 143.0 - 190.9 g
- Housing: the rats were housed individually in Polycarbonate cages type III (floor area about 800 cm²)
- Diet (e.g. ad libitum): Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland.
- Water (e.g. ad libitum): potable tap water in water bottles
- Acclimation period: The animals were acclimated to the laboratory conditions between start of the study and first administration (GD 6).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 /12

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance solutions were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature.
For the test substance preparations, the specific amount of test substance was weighed, topped up with deionized water in a graduated flask and intensely mixed with a magnetic stirrer until it was completely dissolved.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in deionized water at room temperature over a period of 7 days had been verified prior to the start of the study
Details on mating procedure:
The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”.
Duration of treatment / exposure:
From implantation to one day prior to the expected day of parturition (GD 6-19).
Frequency of treatment:
Once a day
Duration of test:
ON GD20 all surviving dams were sacrified and examined macroscopically.
Dose / conc.:
5 mg/kg bw/day
Dose / conc.:
20 mg/kg bw/day
Dose / conc.:
60 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. During the administration period (GD 6-19) all animals were checked daily for any abnormal clinically signs before administration as well as within 2 hours and within 5 hours after administration.


DETAILED CLINICAL OBSERVATIONS: Yes
The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).

BODY WEIGHT: Yes
All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results.
Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).


FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13- 15, 15-17, 17-19 and 19-20.

POST-MORTEM EXAMINATIONS: Yes / No / No data
On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomized order.
After the dams had been sacrificed, they were necropsied and assessed for gross pathology
Ovaries and uterine content:
Weight of the unopened uterus, Number of corpora lutea, Number and distribution of implantation sites classified as: Live fetuses
Dead implantations:
Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)
Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)
After the weight of the uterus had been determined, all subsequent evaluations of the dams and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.
Fetal examinations:
At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined´ macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus). After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.
Soft tissue examination of the fetuses
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.
Skeletal examination of the fetuses
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually
Statistics:
Means and standard deviations were calculated. In addition the following statistical analysis will be carried out:
Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus,
number of corpora lutea, number ofimplantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight. DUNNETT-Test
Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings. FISHERS EXACT-Test
Proportions of fetuses with malformations, variations and/or unclassified observations in each litter.WILCOXON-Test
Indices:
Conception rate, preimplantation loss, postimplantation loss.
Historical control data:
Historical control data were available
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights of the low-, mid- and high-dose dams (5, 20 or 60 mg/kg bw/d) were in general comparable to the concurrent
control group throughout the entire study period. Consistent to the reduced food consumption value from GD 6 to 8, the average body
weightgain of the dams in test group 3 (60 mg/kg bw/d) was statistically significantly decreased onGD 6-8. During GD 6-8 10 out of 25 animals showed a body weight loss (up to -15.3 g) which recovered during GD 8-10 and thereafter. If calculated for the entire treatment period (GD 6-19) or the entire study period (GD 0-20), the mean body weight gain of the high-dose damswas comparable to the
concurrent control group. The decreased body weight gain of the highdosedams during GD 6-8 was considered to be treatment-related and adverse.
The average body weight gain of the low- and mid-dose dams (5 or 20 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period.
The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) was distinctly and statistically significantly lower in test group 3 (60 mg/kg bw/d - about 24% below the concurrent control value).
Furthermore, the carcass weight of the high-dose dams was also reduced (without attaining statistical significance, about 3% below controls). The decrease in corrected body weight gain was considered to be treatment-related and adverse.
The corrected body weight gain of test groups 1 and 2 (5 and 20 mg/kg bw/d) revealed no difference of any biological relevance to
the corresponding control group. Moreover, mean carcass weights remained also unaffected by the treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The mean food consumption of the dams in test group 3 (60 mg/kg bw/d) was statistically significantly decreased on GD 6-8 (about 17% below control), but recovered afterwards and was comparable to the concurrent control group throughout the remaining study period (GD 8- 20). The decrease showed a high standard deviation value which might be caused by two individuals with very low food consumption values. However, if calculated for the entire treatment period (GD 6-19) or study period (GD 0-20), the mean food consumption of the high-dose dams was generally comparable to the concurrent control. Therefore, the reduced food consumption value during GD 6-8 was considered to be spontaneous and not treatment-related.
The mean food consumption of the dams in test groups 1 and 2 (5 and 20 mg/kg bw/d) was quite similar to the concurrent control throughout the entire study period.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At gestation day 20 in dams of test group 3 (60 mg/kg bw/d) red blood cell (RBC) counts, hemoglobin, hematocrit and mean
corpuscular hemoglobin concentration (MCHC) values were significantly decreased and absolute reticulocyte counts were increased.
Absolute reticulocyte counts were already statistically significantly higher in dams of test group 2 (20 mg/kg bw/d), but this was an isolated alteration of the red blood cell parameters and the mean was within the historical control range (absolute reticulocytes 77.4 – 187.0 giga/L). Therefore, increased absolute reticulocyte counts in dams of test group 2 were regarded as incidental and not
treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
At gestation day 20, in dams of test group 3 (60 mg/kg bw/d) glucose and total bilirubin values were significantly increased and
creatinine values were significantly decreased. However, all means were within historical control ranges (glucose 4.76-5.81 mmol/L;
total bilirubin 0.67-1.66 μmol/L; creatinine 22.8-31.5 μmol/L). Therefore, all mentioned alterations were regarded as incidental and not
treatment-related.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Animals of all test groups (1-3) showed significantly increased relative liver weights, in animals of test groups 2 (20 mg/kg bw/d) and
3 (60 mg/kg bw/d) absolute liver weights were increased in addition. The increased absolute (+111%) and relative (+114%) liver weights of test group 3 animals and increased absolute (+8%) and relative (+9%) liver weights of test group 2 animals were assumed as
treatment related. A relation to treatment of the very slightly increase relative liver weights (+4%) in test group 1 animals is rather
unlikely, since only relative weight parameters were significantly changed and since the weight increase was very slight.
The increased absolute and relative weight of the spleen in test group 3 animals were assumed as treatment related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The duodenal wall was thickened in 13/25 animals of test group 3 (60 mg/kg bw/d) and 2/25 animals of test group 2 (20 mg/kg bw/d).
The thickening of duodenal wall was assumed as treatment related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Details on maternal toxic effects:
For more details cf. "Attached background material"
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
haematology
gross pathology
Fetal body weight changes:
no effects observed
Changes in sex ratio:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Only one low-dose fetus with multiple external malformations was seen: female fetus No. 47- 10 (5 mg/kg bw/d) had an exophthalmus on its left eye, micrognathia and microstomia. These external findings were associated with multiple skeletal malformations concerning the skull.
The overall incidences of external malformations were comparable to those found in the historical control data and, therefore, assessed as incidental.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Individual fetuses of test groups 1-3 (5, 20 or 60 mg/kg bw/d) had skeletal malformations. One low-dose fetus had associated external malformations. The finding ‘misshapentuberositas deltoidea’ was observed in one single fetus of test groups 2 and 3 each.
However, the finding can be found in the historical control data in a higher incidence and wasassessed as not treatment-related.
The number of these malformations adds up to a statisticallysignificantly higher value for the total skeletal malformation rate in the mid-dose group(mean of affected fetuses/litter: 2.3). However, the values are clearly inside the historical controlrange (HCD of affected fetuses/litter: 0.8%, andshowed no dose-dependency. Therefore, it was assessed as not treatment-related.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Three soft tissue variations were detected in all test groups including the control (0, 5, 20 or 60 mg/kg bw/d), i.e. short innominate, dilated renal pelvis and dilated ureter. The incidences of these variations were neither statistically significantly different from control nor dose-dependent and, therefore, not considered biologically relevant. All of them can be found in the historical control data at comparable incidences.

Fetal skeletal unclassified cartilage obvervations
Additionally, some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all test groups.. The observed unclassified cartilage findings were related to the skull, the ribs and
the sternum and, in most cases, did not show any relation to dosing. The incidence of ‘branched rib cartilage’ was statistically
significantly increased in test groups 1-3 (2.5%* / 3.7%** / 3.7%** affected fetuses per litter versus 0.0% in control) but was well within the historical control range (HCD of affected fetuses per litter: 2.1% [0.0 - 6.0]). Therefore, it was not assessed as treatment-related.
The overall incidences of skeletal unclassified cartilage observations in the substance- treated groups did not differ significantly from
the concurrent control group.
Details on embryotoxic / teratogenic effects:
External, soft tissue and skeletal malformations were noted in all test groups (0, 5, 20 and 60 mg/kg bw/d). Some fetuses were multiple malformed: one male control fetus had a situs inversus combined with an abnormal lung lobation. For one male fetus (5 mg/kg bw/d) severely malformed cervical vertebrae were recorded. The findings in one female fetus (5 mg/kg bw/d) consisted of multiple external malformations (exophthalmus, micrognathia, microstomia), comprising
severely malformed skull bones, while another male fetus (20 mg/kg bw/d) had a severely malformed vertebral column and/or ribs. No ontogenetic pattern is recognizable for the individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of the low-, mid- and high-dose groups. All of them are present in the historical control data of the rat strain. Thus, a relationship of these four cases of malformed fetuses to the treatment is not assumed. Three malformations, i.e. split scapula, malpositioned and bipartite sternebra and misshapen tuberositas deltoidea, were not related to dose and most of them can be found in the historical control data. An association of these findings to the treatment is also not assumed. The total number of the fetal skeletal malformations added up to a statistically significantly higher value in the mid-dose group (mean of affected fetuses/litter: 2.3) compared to control.
However, the values were clearly inside the historical control range (HCD of affected fetuses/litter: 0.8%, [0.0 - 3.0]; and showed no dose-dependency. Therefore,
it was assessed as not treatment-related and not adverse.
External variations did not occur in any of the fetuses of this study. Some soft tissue variations and a range of skeletal variations occurred in all test groups including the controls. None of the total incidences showed a relation to dosing. The majority of individual variations were equally distributed about the different
test groups, if normal biological variation is taken into account, and can be found in the historical control data at a comparable frequency.
One skeletal variation – wavy ribs - was dose-related increased without statistical significance and, in the high dose group, above the historical control range (mean of affected fetuses per litter: 11.0 %, HCD of affected fetuses per litter: 4.6% [0.8 - 8.7]). Wavy ribs were noted in 15 pups out of 11 litters (test group 3, 60 mg/kg bw/d). Wavy ribs represent a common finding in prenatal toxicity studies. Numerous studies have been conducted to study the reversibility of wavy ribs during postnatal development. They have shown that wavy ribs represent a reversible finding. The finding represents minor departures from normal morphology
or slight delays of ossification, which did not affect morphology. In the high-dose group they occur in the presence of maternal toxicity. Thus, the marginal increase of the finding above the background rate of the used rat strain is considered neither as adverse nor of toxicological relevance.
No unclassified external and no unclassified soft tissue observations were recorded for any of the fetuses in this study. A spontaneous origin is assumed for the unclassified skeletal cartilage observations which were observed in several fetuses of all test groups (0, 5, 20 and 60 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment.
The incidence of ‘branched rib cartilage’ was statistically significantly increased in test groups 1-3 (2.5%* / 3.7%** / 3.7%** affected fetuses per litter versus 0.0% in control) but was well within the historical control range (HCD: 2.1% [0.0 - 6.0]). Therefore, it was not assessed as a treatment-related, adverse event. The overall incidences of skeletal unclassified cartilage observations in the substance-treated groups did not differ significantly from the concurrent control group.
Finally, fetal examinations revealed that there is no effect of the compound on the respective morphological structures up to a dose of 60 mg/kg bw/d.
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Abnormalities:
not specified
Developmental effects observed:
not specified

For more details cf. "Attached background material"

Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of 3- Hexyne-2,5-diol to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused evidence of maternal toxicity in the mid- and high-dose group (20 and 60 mg/kg bw/d), such as decreased (corrected net) body weight gain, a normocytic, hypochromic, regenerative anemia based on altered hematology parameters (associated with increased spleen weights at 60 mg/kg bw/d), and thickening of duodenal wall in pathology. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 5 mg/kg bw/d.
Under the conditions of the present study the test substance is not teratogenic in rats. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 60 mg/kg bw/d, the highest dose tested.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and guideline compliant study.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a prenatal developmental toxicity study (BASF SE, 2017) the test substance was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle. Generally, clinical observations including food consumption revealed no toxicologically relevant differences between the animals receiving 5, 20 and 60 mg/kg bw/d 3-Hexyne-2,5-diol and controls. Body weight (change) of dams of low and mid dose group showed no treatment-related, adverse findings. In the high-dose dams (test group 3, 60 mg/kg bw/d), a statistical significant decrease of body weight gain during GD 6-8 with a body weight loss in 10 out of 25 dams and of corrected net body weight gain (24 % below control) was observed. These findings were assessed as treatment-related and adverse. No differences of toxicological relevance between the control and the treated groups were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Regarding clinical pathology, a normocytic, hypochromic, regenerative anemia occurred in dams of test group 3 (60 mg/kg bw/d). This was indicated by decreased red blood cell (RBC) counts, hemoglobin and hematocrit values as well as decreased mean corpuscular hemoglobin concentrations (MCHC) and increased absolute reticulocyte counts. It was assessed as treatment-related and adverse. Regarding pathology, target organs were the liver, spleen and duodenum. In animals of test groups 2 (20 mg/kg bw/d) and 3 (60 mg/kg bw/d) the significantly increased absolute and relative liver weights were assumed as treatment related but non-adverse, since the weight increase was only mild and all liver parameters in clinical chemistry examination were within the normal range. Thus, there was no impact on the functionality of the liver. Histopathology was not performed. The increased absolute and relative weights of the spleen in test group 3 animals are assumed to be a treatment related and adverse finding, since the hematological examination revealed a regenerative anemia which is likely associated to increased spleen weights. Histopathology was not performed.

Macroscopically, the duodenal wall was thickened in 13/25 animals of test group 3 (60 mg/kg bw/d) and 2/25 animals of test group 2 (20 mg/kg bw/d). The thickening of duodenal wall was regarded as treatment related. Without histopathology, the nature of this finding cannot be elucidated and, therefore, precautional assessed as adverse.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

No influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose. Fetal external, soft tissue and skeletal examinations revealed that there is no effect of the compound on the respective morphological structures up to a dose of 60 mg/kg bw/d.

Under the conditions of this prenatal developmental toxicity study, the oral administration of 3-Hexyne-2,5-diol to pregnant Wistar rats from implantation to one day prior to the expected day

of parturition (GD 6-19) caused evidence of maternal toxicity in the mid- and high-dose group(20 and 60 mg/kg bw/d), such as decreased (corrected net) body weight gain, a normocytic,

hypochromic, regenerative anemia based on altered hematology parameters (associated withincreased spleen weights at 60 mg/kg bw/d), and thickening of duodenal wall in pathology. In

conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 5 mg/kg bw/d.

Under the conditions of the present study the test substance is not teratogenic in rats. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 60 mg/kg

bw/d, the highest dose tested.

Justification for classification or non-classification

The available data do not trigger classification or labelling for effects on fertility and developmental toxicity according to Directive 67/548/EEC (DSD) and EU Classificaton,Labelling and Packaging of Substances and Mixtures Regulation (EC) No 1272/2008 (CLP).