2,4,7,9-tetramethyldec-5-yne-4,7-diol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

2,4,7,9-Tetramethyl-5-decyne-4,7-diol, when fed to rats under the conditions of an one generation reproductive toxicity study, showed no effect at 500 mg/kg/day.

At dose levels at greater than or equal to 1,000 mg/kg/day slightly decreased mean weaning weights, lacation indices and body weight gain were observed in the F1a generation.

There were no significant effects on the reproductive performance reported.

Additionally, based on the lack of effects on F0 reproductive parameters and F1 neonatal survival and growth in an OECD 422 study, 7500 ppm (highest concentration tested) was considered to be the NOAEL for F0 reproductive toxicity and F1 neonatal toxicity. This concentration corresponded to mean dose levels of 518 and 633 mg/kg/day for F0 males and females during the premating period, Gestation Days 0–20, Lactation Days 1–4, and Lactation Days 4-13, respectively.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

other: one generation reproduction study preceeds OECD Guidelines

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

not applicable

Principles of method if other than guideline:

Design of Study I Single Generation Reproduction Study in the Rat (F0->F1a):
Design of study II Ninety One Day Feeding Study (F1a Rats):

Ten male and twenty female sexually mature rats per groups were exposed to the following levels of 2,4,7,9-Tetramethyl-5-decyne-4,7-diol:

1. Low Dose: 500 mg/kg/d
2. Mid Dose: 1,000 mg/kg/d
3. High Dose: 2,000 mg/kg/d

The animals were mated and the newborn raised to weanling age.
The weanlings were randomized to their respective groups according to SOPs and carried on the same levels to the termination of the experiment.
Body weight and feed consumption data as well as several reproductive parameters were taken from the Fo rats.
Body weight and consumption data were taken weekly from the F1a rats.
Certain hematological and urine analytical parameters were performed on five male and five female F1a rats per group after 45 days and after 91 days.
Certain clinical chemistry parameters were performed on five male and five female rats per group after 91 days on test.
Gross necropsy was performed on all rats.
Organ weights and organ-to-body weight ratios were done on ten male and ten female F1a rats per group.
Complete histopathology was done on ten male and ten female rats from the high dose and control group while the major organs were examined histopathologically from all remaining survivors.

GLP compliance:

no

Remarks:

study performed before implementation of GLP

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass.
- Acclimation period: four weeks
- Housing: The animals were housed individually during quarantine in steel wire mesh suspended cages in a room by themselves with a constant temperature of 73° +/- 3° F (equal to 23° +/- 2° C), with a constant humidity of approximately 40 % to 70 % and with 100 % fresh air make-up, approximately eight to ten complete air changes per hour.
- Diet, Water: Feed and water were provided ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23° +/- 2° C
- Humidity (%): 40 % to 70 %
- Air changes (per hr): 8 - 10

Route of administration:

oral: feed

Vehicle:

other: diet: Charles River 19RF, Rat, Mouse, Hamster Meal

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION

- Rate of preparation of diet (frequency):
The test diets were prepared weekly by the respective study coordinator, based on the mean body weight and the mean feed consumption during the second previous week.

- Mixing appropriate amounts with (Type of food):
The basic feed supply for the duration of the experiment was "Charles River 19RF, Rat, Mouse, Hamster Meal" manufactured by Agway of syracuse, New York. Test diets were prepared weekly by mixing the appropriate amount of 2,4,7,9-Tetramethyl-5-decyne-4,7-diol with the appropriate amount of basic diet in a three cubic feet Patterson Kelley twin shell mixer, tumbling at a rate of 40 tumples per minute around a high speed coaxial mixing bar equipped with discs bearing slanted blades rotating at the rate of 2,000 rpm. Fresh diets were prepared weekly. Feed consumption was measured by weighing the feed containers full of feed at the beginning of the week (full weight) and the same feed containers with the remaining feed at the end of the week (empty weight). All feed remaining at the end of the week was disposed of.

Details on mating procedure:

- M/F ratio per cage: individual housing

Analytical verification of doses or concentrations:

not specified

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Basis:
nominal in diet

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Basis:
nominal in diet

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Basis:
nominal in diet

No. of animals per sex per dose:

10 male
20 female

Control animals:

yes

Clinical signs:

no effects observed

Description (incidence and severity):

No rats expired in this phase of the experiment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Only one pertinent finding: The mean body weight of the high dose female group after weaning its young was significantly lower than the corresponding control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Only one pertinent finding: The mean body weight of the high dose female group after weaning its young was significantly lower than the corresponding control.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathology examination of the reproductive organs from all Fo parents revealed no abnormalities.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: The feed consumption of the high dose female group after weaning its young was significantly lower than the control. There was no other pertinent findings.

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): All rats survived for the duration of the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): Only one pertinent finding: The mean body weight of the high dose female group after weaning its young was significantly lower than the corresponding control.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): The feed consumption of the high dose female group after weaning its young was significantly lower than the control. There was no other pertinent findings.


GROSS PATHOLOGY (PARENTAL ANIMALS): No gross abnormalities were observed in any of the Fo parents, either male or female.

HISTOPATHOLOGY (PARENTAL ANIMALS): Histopathology examination of the reproductive organs from all Fo parents revealed no abnormalities.

OTHER FINDINGS (PARENTAL ANIMALS): None

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormal clinical sign were observed in any of the rats, either test or control, during the entire study period.

Mortality / viability:

no mortality observed

Description (incidence and severity):

No rats expired in this phase of the experiment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was persistent statistically significant decrease in the rate of mean weekly body weight gain in the high dose rats, goth male and female, througout the experiment, for most weeks in the mid dose rats, both male and female, and for ...

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increase of liver weight

Gross pathological findings:

no effects observed

Description (incidence and severity):

No pertinent gross pathology findings were observed in any of the F1a rats at terminal sacrifice.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

changes in the structure of the liver due to the chronic exposure to the test material

VIABILITY (OFFSPRING): No rats expired in this phase of the experiment.

CLINICAL SIGNS (OFFSPRING): No abnormal clinical sign were observed in any of the rats, either test or control, during the entire study period.

BODY WEIGHT (OFFSPRING): There was persistent statistically significant decrease in the rate of mean weekly body weight gain in the high dose rats, goth male and female, througout the experiment, for most weeks in the mid dose rats, both male and female, and for a few weeks in the low dose male rats. The body weight in all the test groups was significantly lower from the corresponding control groups even during zero week due to the fact that these animals came from dams already on various levels of the compound. The rats in the various groups at zero week had widely variable initial body weights because the corresponding Fo dams conceived - and gave birth - at different times.The difference between either the low dose groups and the control groups or the mid dose groups and the control groups is less than 10 % of the corresponding control weight during the last six weeks on test while both high dose groups differed by mor than 10 % from the beginning. Because of this factor we do not consider the low dose and the mid dose difference to be biologically significant.

SEXUAL MATURATION (OFFSPRING)

ORGAN WEIGHTS (OFFSPRING):
Heart: The mean cardiac weight of the high dose male rats was significantly lower than the control. However, there was no morphologic abnormalities observerd microscopically.
Liver: The mean liver weight of the mid and high dose male rats, as well as of all test female rats was significantly higher than the corresponding control rats.
Kidneys: The mean renal weights were not remarkable.
Gonads: The mean testicular weights were not remarkable, while the ovarian weight of the mid dose female group was significantly less than the control group. There was no accompanying histopathology, however, of the ovaries of the mid dose rats.
Brain: The mean brain weight of all the male test groups and of the high dose female group was significantly lower than the corresponding control weight. We do not consider this, hoever, to be of any biological significance since there was no accompanying histopathology of the brain.

GROSS PATHOLOGY (OFFSPRING): No pertinent gross pathology findings were observed in any of the Fla rats at terminal sacrifice.

HISTOPATHOLOGY (OFFSPRING): The most frequent finding was lymphocytosis of the lungs, either peribronchial or perivascular. This condition, endemic in this strain of rat, was seen in the control as well as in the test animals. The most significant finding, however, was the cloudy swelling of the liver seen in a dose dependent way in the mid dose and high dose rats, both male and female. This most likely implies an adaptive hyperplasia of the sub-cellular endoplasmic reticulum of the hepatic cells of the affected test groups.

OTHER FINDINGS (OFFSPRING):

Organ-to-body weight ratios:
Heart-to-body weight ratios: These were not remarkable.
Liver-to-body weight ratios: The ratios for both male and female test groups were considerably higher than the corresponding control values with a dose dependent pattern. The female ratios were slightly higher the the male ones.
Kidneys-to-body weight ratios: These were not remarkable.
Gonads-to-body weight ratios: These were not remarkable.
Brain-to-body weight ratios: These were not remarkable.

Clinical Chemistry:
FBS: Low dose female rat #1070 had an FBS of 260 mg/dl; histopathologically this rat was not remarkable.
BUN: All values, individual as well as mean, were normal.
SGOT: All values, individual as well as mean, were normal.
SGPT: The mean mid dose female and doth high dose male and female values were significantly higher than control. These differences are of no biological significance, however, since all mean as well as individual values except one were normal; high dose male rate #1117 had an SGPT value of 105. Histopathologically this rat exhibited mild pulmonary lymphocytosis.
GGTP: All values, individual as well as mean, were normal.
Total Protein: The mean value for the mid dose groups, male and female, as well as for the high dose groups, both male and female, was significantly higher than control. All mean as well as individual values except one were normal; mid dose female rat #1028 had a Total Protein value of 8.52 g/dl. Histopathologically this rat exhibited pulmonary lymphocytosis.

Hematology:
Hematological Value at 45 Days:
RBC: The mean total RBC counts in the mid dose and the high dose male test groups were significantly lower than the control. All values, however, mean as well as individual, were within our normal range.
HGB: The mean hemoglobin values of the mid dose and high dose male rats were significantly lower than the control, while the same value was higher than the control in the high dose female rats. All values, however, mean as well as individual, were within our established normal range.
HMCT: The mean HMCT values for the low and high dose group of male rats were statistically lower than the control, while the mean HMCT value for the high dose female group was statistically higher than the control. All values, however, mean as well as individual, were within normal range.
WBC Diff: All rats examined displayed normal differential counts.
Plat. Est.: The platelets of all rats examined were deemed to have been adequate.

Hematological Values at 91 Days:
RBC: All total RBC counts, as well as individual, were within normal range.
HGB: All HGB values, mean as well as individual, except one were within normal range; high dose female rat #1122 had a HGB of 11.6 g/dl; histologically this rat exhibited mild pulmonary lymphocytosis.
HMCT: All HMCT values, mean as well as individual, were within normal range.
WBC: The mean total WBC counts of the low and high dose groups were significantly higher than the control groups. All total WBC counts, however, mean as well as individual, were within normal range.
WBC Diff: All rats examined displayed a normal differential counts.
Plat. Est.: The platelet estimate of all rats examined were deemed to have been adequate.

Urinalysis:
Urinalysis at 45 days: There were no significant differences between the test and control groups, male or female, in any urinalysis parameters.
Urinalysis at 91 days: There were no significant differences between the test and control groups, male or female, in any urinalysis parameters.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

haematology

histopathology: non-neoplastic

Critical effects observed:

no

Reproductive effects observed:

no

The only pertinent findings observed in the F0 parents were:
a slight decrease in the mean weaning weight of both male and female pups of the high-dose group, a slight decrease in lactation indices of the high-dose group, decreased body weight and feed consumption of the high-dose female group and normal histology of the reproductive organs in the F0 parents. Fertility, viability and gestation indices were not affected.  In the reproduction phase of this experiment
there was a toxic effect at the 2,000 mg/kg/day level, a borderline effect at the 1,000 mg/kg/day level and no effect at 500 mg/kg/day.

Test item: Summary of F0/F1a Reproduction Data

Dose Group         Number of Dams                  Total Number of Pups
(mg/kg/day)        Mated        Conceived        Live-         Still   Day 4        Culled  
Day 21                     
                                        born    born        
   Day 4*
0                 20          20                 235          4        226          43          180
500                 20          20                 252          2        247          51          195
1000                 20          20                 229          0        220          37          173
2000                 20          19                 225          4        214          27          164
* Litters with more than 10 pups were culled to 10

Surfynol 104: Summary of F0/F1a Reproduction Data
(continued)
Dose Group    Avg. # of Pups/Litter          Avg. Pup
Weaning Weight (g)
(mg/kg/day)   Day 4           Weaned 
              M      F            M      F              Male        Female
0             6.0    5.4            4.6          4.5              45.7        44.2
500             6.6    5.8            5.0          4.8              42.0        40.9
1000             5.8    5.3            4.6          4.1              36.6        35.8
2000             5.5    5.8            4.1          4.6              28.8        25.7
The following pertinent findings were observed in the F1a rats: slight decrease in the mean rate of body weight gain in both sexes at the mid- and high-dose (there was also a significant decrease in this parameter in the low-dose male group during the first eight weeks), normal mean hematological findings, clinical chemistry findings, and urinalysis findings after 91 days on test, significant increase in the absolute and relative liver weights of both sexes at the mid- and high-dose, corresponding histopathology of the liver showing mild to moderate centrilobular cloudy swelling of hepatocytes of the mid- and high-dose rats.  Surfynol 104, when fed to rats under the conditions of this experiment, showed no effect at
500mg/kg/day but did have a toxic effect in the Fla generation at >1,000 mg/kg/day.

Conclusions:

2,4,7,9-Tetramethyl-5-decyne-4,7-diol, when fed to rats under the conditions of an one generation reproductive toxicity study, showed no effect at 500 mg/kg/day.
At dose levels at greater than or equal to 1,000 mg/kg/day slightly decreased mean weaning weights, lacation indices and body weight gain were observed in the F1a generation.
There were no significant effects on the reproductive performance reported.

Executive summary:

The purpose of this study was to evaluate the possible toxicity of the test item, when fed to the rat during a single generation reproduction study and for ninety one days to the F1a weanlings. The test material was mixed into the rats’ feed to provide dose levels of 0, 500, 1000, and 2000 mg/kg/day. Sexually mature Sprague-Dawley albino rats were divided into four groups, each consisting of ten male and twenty female rats. All F0 male rats, both test and control, were fed their respective diets until their litters reached the age of 21 days for weaning, when the F0 dams were sacrificed. The weanlings were randomized to their respective groups and carried on the same dose levels to the termination of the experiment. The only pertinent findings observed in the F0 parents were: 1. Slight decrease in the mean weaning weight of both male and female pups of the high-dose group, 2. Slight decrease in lactation indices of the high-dose group, 3. Decreased body weight and feed consumption of the high-dose female group, 4. Normal histology of the reproductive organs in the F0 parents. The following pertinent findings were observed in the F1a rats: 1. Slight decrease in the mean rate of body weight gain in the mid- and high-dose male and female rats; there was also significant decrease in this parameter in the low-dose male group during the first eight weeks, 2. Normal mean hematological findings, clinical chemistry findings, and urinalysis findings after 91 days on test, 3. Significant increase in the liver weight of the mid- and high-dose male and female test groups with corresponding increase in the liver-to-body weight ratios, 4. Corresponding histopathology of the liver of the mid- and high-dose male and female rats, showing mild to moderate centrilobular cloudy swelling of hepatocytes.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The one-generation reproductive toxicitx study was performed before GLP and Guidelines were established: Klimisch rating 2.
The OECD 422 study is reliable without restrictions.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The substance has no structural alerts for reprotoxicity. This one-generation reproductive toxicity study contains data which are considered to be sufficient to meet the criteria set forth in Regulation EC No. 440/2008 for filling REACH endpoints. The data is generated in a reliable laboratory using established protocols and the results are supported by a fully reliable OECD 422 study.

Effects on developmental toxicity

Description of key information

An OECD 414 study was performed with 2,4,7,9-Tetramethyl-5-dcyne-4,7-diol. There were no adverse maternal effects or adverse effects on intrauterine growth and survival or fetal morphology by test substance exposure up to 15,000 ppm. Based on these results, an exposure level of 15,000 ppm equivalent to 1058 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and for prenatal developmental toxicity when 2,4,7,9-Tetramethyl-5-decyne-4,7-diol was administered orally in the diet to time-mated Crl:CD(SD) rats.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan - Sept 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Females (if applicable) nulliparous and non-pregnant: Naïve, purpose-bred, time-mated (Day mating is confirmed [copulatory plug] = Gestation Day [GD] 0)
- Age at study initiation: 10 to 13 weeks
- Weight at study initiation: 175 to 300 g
- Housing: Single/Individual; Individual housing of presumed pregnant females is required to adequately monitor the health of these females by allowing collection of individual food consumption and appropriate identification of cage observations in the event of early delivery .
Solid-bottom cages containing appropriate bedding material (Bed-OCobs ® or other suitable material). Nesting material will not be provided, as euthanasia is scheduled prior to anticipated parturition. For enrichment, animals will be provided items such as treats and/or a gnawing device, except when interrupted by study procedures/activities.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, ad libitum
- Water (e.g. ad libitum): Municipal tap water, treated by reverse osmosis and ultraviolet irradiation, ad libitum
- Acclimation period: Time-mated rats were received by Charles River on Gestation Day 1, 2, 3 and 4. The day on which confirmation of mating is made was designated Gestation Day 0. Each rat was inspected by a qualified technician upon receipt. All rats were permanently identified with a microchip.
Each rat was observed for mortality/moribundity twice daily. Cage-side observations were performed daily from receipt until Gestation Day 6 (except on days that detailed clinical bservations are performed). Prior to the start of dose administration, those animals judged to be suitable test subjects were identified. Detailed clinical observations and body weights on gestation Days 5 and 6, and food consumption from Gestation Day 5-6 were recorded for all rats (including alternates purchased for the study). The Gestation Day 0 body weight of each dam was recorded by the supplier.

DETAILS OF FOOD AND WATER QUALITY:
Diet: Results of analysis for nutritional components and environmental contaminants are provided by the supplier and are on file at the Testing Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
Water: Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility. It is considered that there are no known contaminants in the water that would interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C to 25°C
- Humidity (%): 30% to 70%
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN-LIFE DATES:
From: 21. Jan. To: 11. Feb. 2022

Route of administration:

oral: feed

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): PMI Nutrition International, LLC LabDiet Certified Rodent Diet 5002
- Storage temperature of food: 18-24°C

Analytical verification of doses or concentrations:

yes

Remarks:

by gas chromatography (GC) with flame ionization detection (FID)

Details on analytical verification of doses or concentrations:

Diet formulation samples were collected for analysis weekly for analysis of homogeneity and concentration.
Dose analysis results were verified prior to diet administration at each sampling interval, if
possible. If results are deemed unacceptable, the diet formulations will be prepared again and
analyzed.

Details on mating procedure:

n.a.; females were purpose-bred, time-mated

Duration of treatment / exposure:

Gestation Days 6 through 2

Frequency of treatment:

continuously

Duration of test:

Gestation Days 0 through 21

Dose / conc.:

0 ppm

Dose / conc.:

1 500 ppm

Remarks:

in diet, corresponding to 109 mg/kg bw/d

Dose / conc.:

5 000 ppm

Remarks:

in diet, corresponding to 354 mg/kg bw/d

Dose / conc.:

15 000 ppm

Remarks:

in diet corresponding to 1058 mg/kg bw/d

No. of animals per sex per dose:

22

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on results dose range finding study
- Rationale for animal assignment (if not random): n.a.
- Fasting period before blood sampling for (rat) dam thyroid hormones: none
- Time of day for (rat) dam blood sampling: prior to noon

Maternal examinations:

- Mortality: all animals; at least twice daily
- Detailed clinical observations: all animals; Gestation Days 5, 6, 9, 12, 15, 18, and 21; Mortality and all signs of overt toxicity were recorded on the day observed. The observations shall include, but are not limited to, evaluations for changes in appearance of skin and fur, eyes, mucous membranes, respiratory and circulatory system, autonomic and central nervous systems, somatomotor activity, and behavior.
- Cage-side observations: Once daily
- Individual body weights: all animals; Gestation Days 0 (by supplier), 5, 6, 9, 12, 15, 18 and 21
- Food consumption: all animals; Gestation Days 5, 6, 9, 12, 15, 18, and 21
- Thyroid hormone assessment: all animals on Gestation Day 21(T3, T4 and TSH)
- Macroscopic examination: all animals on Gestationd Day 21; The cranial, thoracic, abdominal, and pelvic cavities will be opened and the contents examined.
- Organ weights: uterus, thyroid gland and liver
- Microscopy: all animals; thyroid gland

Ovaries and uterine content:

The uterus of each female were excised and its adnexa trimmed. Corpora lutea were also counted and recorded.
The uterus of each female was opened and the number of viable and nonviable fetuses, early and late resorptions and total number of implantation sites was recorded, and the placentae were examined. The individual uterine distribution was documented using the following procedure: all implantation sites, including early and late resorptions, were numbered in consecutive fashion beginning with the left distal uterine horn, noting the position of the cervix and continuing from the proximal to the distal right uterine horn. Uteri which appear nongravid by macroscopic examination were opened and placed in a 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).

Fetal examinations:

External, internal, and skeletal fetal findings were recorded 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), malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life), or incidental (minor changes in coloration, mechanical damage to specimen, etc.).

- External:
Each viable fetus was examined in detail, sexed, weighed, tagged, and euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. Anogenital distance was measured for all viable fetuses. The absolute and normalized (relative to the cube root of fetal body weight) values were reported. Nonviable fetuses (the degree of autolysis is minimal or absent) were examined, crown-rump length measured, weighed, sexed, and tagged individually. The crown-rump length of late resorptions (advanced degree of autolysis) were measured, the degree of autolysis recorded, a gross external examination performed (if possible) and the tissue was discarded.

- Visceral (internal):
Approximately one-half of the fetuses in each litter was examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (Stuckhardt and Poppe, 1984). This examination includes the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development (Woo and Hoar, 1972). The heads from these fetuses were removed and placed in Harrison's fixative for subsequent processing and soft-tissue examination using the Wilson sectioning technique (Wilson, 1965). Following examination, the carcasses and cephalic slices were discarded. The sex of all fetuses wasconfirmed by internal examination. The remaining fetuses (approximately one-half from each litter, excluding any carcasses without heads) were eviscerated and fixed in 100% ethyl alcohol for subsequent examination of skeletons.

- Skeletal:
Following fixation in alcohol, each eviscerated fetus was macerated in potassium hydroxide and stained with Alizarin Red S and Alcian Blue by a method similar to that described by Dawson (Dawson, 1926) and Inouye (Inouye, 1976). The skeletal examination was made following this procedure.

Statistics:

Descriptive Statistical Analyses: Means, standard deviations, percentages, numbers, and/or incidences were reported, as appropriate by dataset.
Inferential Statistical Methods: All statistical tests were conducted at the 5% significance level. All pairwise comparisons were be conducted using two sided tests and will be reported at the 1% and 5% levels.
Parametric/Non-parametric: Levene’s test was used to assess the homogeneity of group variances.
The groups were compared using an overall one-way ANOVA F-test if Levene’s test is not
significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis
test is found to be significant, then pairwise comparisons were conducted using Dunnett’s or
Dunn’s test, respectively.
Non-Parametric: The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test is found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.
Group Pair-wise Comparison (General ANOVA)

Indices:

Body Weight Gains: Calculated between each scheduled interval as well as between the following intervals: GD 6-21
Food Consumption: Calculated for each corresponding body weight interval
Pre-Implantation Loss = (No. of corpora lutea – no. of implants x 100) / (No. of corpora lutea)
Post-Implantation Loss = (No. of implants – no. of live fetuses x 100) / (No. of implants)
Sex Ratio (% males) = (No. male fetuses x 100) / (Total no. of fetuses)
Litter % of Fetuses with Abnormalities = (No. of fetuses in litter with a given finding x 100) / (No. of fetuses in litter examined)

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related clinical observations were noted at the detailed clinical observations or cage-side observations at any concentration.

Mortality:

no mortality observed

Description (incidence):

All females in the control, 1500, 5000, and 15,000 ppm groups survived to the scheduled necropsy on Gestation Day 21.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 15,000 ppm group, statistically significantly lower mean body weight gain was noted immediately following the initiation of exposure (Gestation Days 6–9), and was followed by statistically significant higher mean body weight gain during Gestation Days 9–12. Lower mean body weight gains were noted in this group during Gestation Days 15–21; the difference from the control group was statistically significant during Gestation Days 15–18. As a result of the fluctuations, mean body weight gains in the 15,000 ppm group was comparable to the control group when the overall exposure period (Gestation Days 6–21) was evaluated. The effects on mean body weight gains in the 15,000 ppm group were considered test substance-related but nonadverse because it had no impact on the overall body weight gain or mean absolute body weights.
Mean maternal body weights, body weight gains, adjusted body weights, and adjusted body weight gains in the 1500 and 5000 ppm groups and mean gravid uterine weight in the 1500 ppm were unaffected by test substance exposure.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean maternal food consumption and food utilization in the 15,000 ppm group were statistically significantly lower than the control group following the initiation of exposure (Gestation Days 6–9), and higher than or comparable to the control group during Gestation Days 9–15; differences were generally statistically significant. Mean food consumption and utilization in this group were generally comparable to or higher than the control group for the remainder of the exposure period (Gestation Days 15–21), with the following exception. A statistically significantly lower mean food utilization value was noted at 15,000 ppm compared to the control group during Gestation Days 15–18. The initial decrement in food consumption and food utilization at 15,000 ppm corresponded to the lower mean body weight gain during this interval; this change was considered test substance-related but nonadverse in the absence of an effect on absolute body weights. When the entire treatment period (Gestation Days 6–21) was evaluated, mean maternal food consumption and food utilization in this group were comparable to the control group.
Mean maternal food consumption and food utilization in the 1500 and 5000 ppm groups were unaffected by test substance exposure.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

In the 5000 and 15,000 ppm groups, nonadverse test substance-related effects on mean TSH, T3, and T4 concentrations were noted on Gestation Day 21. Lower mean T3 concentrations were noted in these groups compared to the control group (10.3 and 25.7% lower, respectively), and
were below the minimum range of the testing facility`s historical control data (version 2020.02; 221.4 pg/mL). Higher mean T4 concentrations in these groups were also noted compared to the control group (14.8 and 21.3% higher, respectively), but were within testing facility`s historical control data range (10,179.2 to 23,580.0 pg/mL).
Higher mean TSH concentrations were noted compared to the control group (14.0 and 54.5% higher, respectively), and were within testing facility`s historical control data range (1281.0 to 2153.1 pg/mL) at 5000 ppm, but exceeded the maximum value at 15,000 ppm.
This correlated with the nonadverse microscopic findings of hepatocellular hypertrophy in the liver at 5000 and 15,000 ppm and follicular cell hypertrophy and decreased colloid in the thyroid gland at 15,000 ppm. The
mechanism of thyroid gland follicular cell hypertrophy was considered to be enhanced thyroid hormone degradation secondary to test substance-related hepatic microsomal enzyme induction, specifically, induction of uridine diphosphoglucuronosyltransferase (UDP-GT) causing enhanced thyroid hormone metabolism and subsequent increased levels of TSH (Zabka et al., 2011).
In the 1500 ppm group, test substance-related lower mean T3 concentration was noted on Gestation Day 21 compared to the control group (10.2% lower) and was below the minimum range of the testing facility`s historical control data (221.4 pg/mL). This was considered nonadverse because there were no effects on thyroid gland weight or histopathology in the thyroid at this dose level.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related higher mean liver weights in the 5000 and 15,000 ppm group correlated microscopically with hepatocellular hypertrophy and macroscopically with liver enlargement in a single 15,000 ppm group female.
This was considered nonadverse in the absence of adverse microscopic findings.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related enlargement of the liver was observed in a single 15,000 ppm group female, which correlated with higher mean liver weights and hepatocellular hypertrophy noted microscopically. This was considered nonadverse because it was limited to a single animal in the absence of adverse microscopic findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Thyroid follicular cell hypertrophy was characterized by cuboidal follicular cells with vacuolated cytoplasm and occasional nuclear crowding. The finding was often accompanied by small diameter follicles containing decreased colloid. Thyroid gland findings had no effect on mean or individual thyroid gland weights.
Hepatocellular hypertrophy was characterized by large hepatocytes with increased eosinophilic hepatocellular cytoplasm in a centrilobular to panlobular distribution. The finding correlated with higher mean liver weights (absolute and relative to terminal body weight) and enlarged liver observed in a single 15,000 ppm group female.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

not examined

Details on maternal toxic effects:

Intrauterine survival was unaffected by test substance at exposure levels of 1500, 5000, and 15,000 ppm. The mean litter proportion of postimplantation loss (primarily early resorptions) in the 5000 ppm and 15,000 ppm groups (6.40% and 8.95% per litter, respectively) were higher (not statistically significant) when compared to the concurrent control group (3.56% per litter), but was attributed to single animals (42.9% for a 5000 ppm animal and 66.7% for a 15,000 ppm animal) and within the testing facility`s historical control data range (2.61% to 12.60% per litter). Furthermore, the mean numbers of live fetuses in the same respective groups (12.1 and 12.0 per dam,) were comparable to the control group (13.2 per dam) and within the testing facility`s historical control data range (11.44 to 13.72 live fetuses per dam).
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Remarks:

corresponding to 1058 mg/kg bw/d

Basis for effect level:

other: no adverse maternal effects observed

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related effects on intrauterine growth were noted in the 15,000 ppm group. Mean fetal weights (combined, males, and females) were lower (4.18%, 4.15%, and 3.75%, respectively) than the concurrent control group; differences were statistically significant for males and combined sexes and corresponded to the lower mean gravid uterine weight noted in this group. The mean fetal weights in this group were within the testing facility`s historical control data range, and therefore this effect was considered nonadverse. Intrauterine growth was unaffected by test substance at exposure levels of 1500 and 5000 ppm.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not examined

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No test substance-related external developmental variations were observed in fetuses in this study. Findings were limited to single fetuses in the control and 15,000 ppm groups.

Skeletal malformations:

no effects observed

Visceral malformations:

not examined

Description (incidence and severity):

No test substance-related visceral developmental malformations were observed in fetuses in this study.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

The numbers of fetuses (litters) available for morphological evaluation were 237(18), 248(19), 230(19), and 228(19) in the control, 1500, 5000, and 15,000 ppm groups, respectively.
Malformations were observed in 1(1), 1(1), 0(0), and 1(1) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin. When the total malformations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations, when observed in the test substance-treated groups, occurred infrequently, did not occur in a dose-related manner, and/or were within the testing facility's historical control data ranges.
No test substance-related visceral developmental variations were observed in fetuses in this study. Visceral variations were limited to increased incidences of minimal to moderate dilatation of the ureter(s) in the 15,000 ppm group; these variations would not impact postnatal survival, the mean litter proportions were not statistically significantly different from the concurrent control group, and the values were within the testing facility's historical control data range, and therefore were considered the result of biological variability. Other fetal developmental variations, when observed in the test substance-treated groups, occurred infrequently, did not occur in a dose-related manner, and/or were within the testing facility's historical control data ranges.

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Remarks:

corresponding to 1058 mg/kg bw/d

Sex:

male/female

Basis for effect level:

other: no developmental toxic effect observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Treatment related:

not specified

Conclusions:

In conclusion, there were no adverse maternal effects or adverse effects on intrauterine growth and survival or fetal morphology by test substance exposure up to 15,000 ppm. Based on these results, an exposure level of 15,000 ppm equivalent to 1058 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and for prenatal developmental toxicity when 2,4,7,9-Tetramethyl-5-decyne-4,7-diol was administered orally in the diet to time-mated Crl:CD(SD) rats.

Executive summary:

The test substance was administered continuously in the diet during Gestation Days 6–21.

The following parameters and end points were evaluated in this study: mortality, clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, food utilization, thyroid hormones, anogenital distance, organ weights, macroscopic and microscopic examinations, intrauterine growth and survival, and fetal morphology.

Mean calculated test substance consumption during GD 6 -21 (based on analytical determination of diet):

- 0 ppm: 0 mg/kg bw/d

- 1500 ppm: 109 mg/kg bw/d

- 5000 ppm: 354 mg/kg bw/d

- 15000 ppm: 1058 mg/kg bw/d

Test substance-related lower mean body weight gain with correspondingly lower mean food consumption and food utilization were noted in the 15,000 ppm group during Gestation Days 6-9, followed by higher mean body weight gains during Gestation Days 9–12 compared to the control group. Mean body weight gains, food consumption, and food utilization in this group were generally comparable to the control group for the remainder of the treatment period; any transient fluctuations noted did not impact mean absolute body weights. As a result, the initial decrements were considered nonadverse. Lower mean gravid uterine weight was noted in the 15,000 ppm group compared to the control group. The difference was considered test substancerelated because it correlated with lower mean fetal weight at this exposure level but was considered nonadverse because it was within the range of the testing facility's historical control data.

Mean maternal body weights, body weight gains, food consumption, food utilization, and gravid uterine weights in the 1500 and 5000 ppm groups and adjusted body weights and adjusted body weight gains in the 1500, 5000, and 15,000 ppm groups were unaffected by test substance exposure.

Test substance-related nonadverse effects on mean T3 concentrations were noted at all dose levels on Gestation Day 21; lower mean T3 concentrations were noted in these groups compared to the control group. Higher mean T4 and TSH concentrations in the 5000 and 15,000 ppm groups were also noted compared to the control group. This correlated with the nonadverse microscopic findings of hepatocellular hypertrophy in the liver at 5000 and 15,000 ppm and follicular cell hypertrophy and decreased colloid in the thyroid gland at 15,000 ppm. The mechanism of thyroid gland follicular cell hypertrophy was considered to be enhanced thyroid hormone degradation secondary to test substance-related hepatic microsomal enzyme induction, specifically, induction of uridine diphosphoglucuronosyltransferase (UDP GT) causing enhanced thyroid hormone metabolism and subsequent increased levels of TSH (Zabka et al., 2011).

Test substance-related maternal macroscopic findings included enlargement of liver noted in a single 15,000 ppm group female; this correlated with nonadverse hepatocellular hypertrophy noted microscopically in this group and thus was considered nonadverse.

Histopathological examinations revealed adaptive, nonadverse liver and thyroid gland changes. Hepatocellular hypertrophy was noted microscopically in the 5000 and 15,000 ppm groups, which correlated with higher mean liver weights and liver enlargement for a single 15,000 ppm group female. Thyroid gland findings consisted of follicular cell hypertrophy and decreased colloid, with no macroscopic or organ weight correlates.

Test substance-related slightly lower (up to 4.18%) mean fetal body weights (combined, males, and females) were noted in the 15,000 ppm group compared to the control group which corresponded to the lower mean gravid uterine weight noted in this group and were considered nonadverse because they were within the range of the testing facility's historical control data.

Intrauterine growth at 1500 and 5000 ppm and intrauterine survival at 1500, 5000, and 15,000 ppm were unaffected by test substance exposure.

No test substance-related external, visceral, or skeletal developmental malformations or variations were noted at any exposure concentration.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 058 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable without restriction.

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

The substance has no structural alerts for developmental toxicity. 

 

Mode of Action Analysis / Human Relevance Framework

In the absence of any evidence for species specific effects or modes of action the effects observed in animals are regarded as relevant for humans. However, according to latest scientific discussions the relevance of hepatic effects observed in rodents for humans is currently subject of intensive research.

Justification for classification or non-classification

Based on these results and according to CLP (No. 1272/2008 of 16 December 2008), the test item does not have to be classified and has no obligatory labelling requirement for reproductive and developmental toxicity.