2,4,7,9-Tetramethyldec-5-yne-4,7-diol, ethoxylated

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on the lack of effects on F0 reproductive parameters and F1 neonatal survival and growth, a dietary 2,4,7,9-Tetratmethyl-5-decyne-4,7-diol, ethoxylated concentration of 7500 ppm (the highest concentration tested) was considered to be the NOAEL for F0 reproductive toxicity, F0 neurotoxicity, and F1 neonatal toxicity. This concentration corresponded to mean dose levels of 539 and 668 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.

Furthermore, 2,4,7,9-Tetramethyl-5-decyne-4,7-diol and 2,4,7,9-Tetratmethyl-5-decyne-4,7-diol, ethoxylated (the structural analogues), 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.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct. 2021 - Sept 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

Currently, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models that do not use live animals currently do not exist.
This species and strain of rat is recognized as appropriate for reproduction studies. This animal model has been proven to be susceptible to the effects of reproductive toxicants. The number of animals is based on the OECD Guideline for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 29 July 2016.

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

Crl:CD(SD)

Sex:

male/female

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: yes
- Age at study initiation: 10 to 11 weeks
- Weight at study initiation: 225 to 450 g (males)/175 to 300 g (females)
- Housing:
Group housed (2–3 animals of the same sex and same dosing group together) during the premating period.
During cohabitation, the females were paired (1:1) with a male for mating (home cage of the male).
Single/Individual following positive signs of mating or the end of the breeding period.
In Solid-bottom cages containing appropriate bedding material (Bed-OCobs ® or other suitable material). For enrichment, animals were provided items such as treats, a gnawing device, nd/or nesting material, 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: a minimum of 7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C to 26°C
- Humidity (%): 30% to 70%
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark.

IN-LIFE DATES:
From: 12 Oct. To: 21. Dec. 2021

Route of administration:

oral: feed

Vehicle:

other: diet; PMI Nutrition International, LLC LabDiet Certified Rodent Diet 5002

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: -20°C

Details on mating procedure:

Following a minimum of 14 days of exposure, 1 female was cohabited with 1 male rat of the same treatment group, avoiding sibling mating, in a solidbottom cage for mating (home cage of the male). A maximum of 14 days was allowed for mating. If no evidence of mating is obtained after 14 days, the animals were separated without further opportunity for mating, and the female was placed in a solidbottom cage containing bedding material.

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 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.

Duration of treatment / exposure:

Males: for at least 14 days prior to mating and continuing throughout
mating until euthanasia for a minimum of 28 days.

Females: for at least 14 days prior to mating and continuing throughout
mating, gestation and lactation until euthanasia.

Frequency of treatment:

continuously

Details on study schedule:

at least 7 days of acclimatisation
minimum of 14 days pre mating exposure
minimum of 14 days mating period
males. termination of 28 days of exposure
females: exposure during gestation and lactation
culling/first homone analysis of pups: LD 4
temination of females and pups on LD 14

Dose / conc.:

0 ppm

Dose / conc.:

800 ppm

Remarks:

in diet (corresponding to 58/68 mg/kg bw/d for males/females); the test substance was administered as a constant concentration (ppm) in the diet with the following exceptions: The concentration of test substance in the diet was adjusted to 0.5X for females that deliver, during Lactation Day (LD) 4 through euthanasia, to account for approximately 2X higher maternal food consumption (and consequently higher test substance consumption) which is normally associated with
the higher demands of milk production.

Dose / conc.:

2 500 ppm

Remarks:

in diet (corresponding to 184/220 mg/kg bw/d for males/females); the test substance was administered as a constant concentration (ppm) in the diet with the following exceptions: The concentration of test substance in the diet was adjusted to 0.5X for females that deliver, during Lactation Day (LD) 4 through euthanasia, to account for approximately 2X higher maternal food consumption (and consequently higher test substance consumption) which is normally associated with
the higher demands of milk production.

Dose / conc.:

7 500 ppm

Remarks:

in diet (corresponding to 539/668 mg/kg bw/d for males/females); the test substance was administered as a constant concentration (ppm) in the diet with the following exceptions: The concentration of test substance in the diet was adjusted to 0.5X for females that deliver, during Lactation Day (LD) 4 through euthanasia, to account for approximately 2X higher maternal food consumption (and consequently higher test substance consumption) which is normally associated with
the higher demands of milk production.

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on dose range finding study
- Fasting period before blood sampling for clinical biochemistry: overnight
- Post-exposure recovery period in satellite groups: no
- Dose range finding studies: 14 d oral toxicity (diet)

Parental animals: Observations and examinations:

- Mortality: twice daily
- Detailed clinical observations: weekly, beginning on the first day of test diet administration for males through euthanasia, and beginning at the start of estrous lavages for females, until evidence of copulation is observed, or until euthanasia (for females with no evidence of mating).
Gestation Days 0, 4, 7, 11, 14, 17, and 20.
Lactation Days 1, 4, 7, 10, and 13.
Mortality and all signs of overt toxicity will be 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: daily
- Individual body weight:
F0 males: weekly
F0 females: Weekly beginning with the first day of test diet administration until evidence of copulation is observed, or until euthanasia (for females with no evidence of mating).
Gestation Days 0, 4, 7, 11, 14, 17, and 20.
Lactation Days 1, 4, 7, 10, and 13.
- Food consumption:
F0 males: daily
F0 females: Daily, beginning with the first day of test diet administration until the initiation of breeding.
Gestation Days 0-20 (daily).
Lactation Days 1-13 (daily).
Estrous: Daily vaginal lavages performed beginning during the pretest period and continuing through the 2-week premating and mating treatment periods until evidence of mating is observed. Lavaging was continued for those females with no evidence of mating until termination of the mating period.
- Breeding: detection of mating
- Parturition, lactation and littering: Females were observed for dystocia (prolonged or difficult labor) or other difficulties and any findings were recorded. When parturition is judged to be complete, the sex of each pup was determined, pups were examined for gross malformations, and the number of stillbirths and live pups were recorded. Any changes or abnormalities in nesting and nursing behavior were recorded.
- FOB:
males: during the last week (days 24-28); 5/group
females: lactation day 13; 5/group
Home cage observations (Biting, Convulsions/tremors, Feces consistency, Palpebral (eyelid) closure, Posture), handling observations (Ease of handling animal in hand, Ease of removal from cage, Eye prominence, Fur appearance, Lacrimation/chromodacryorrhea, Mucous membranes/eye/ skin color, Muscle tone, Palpebral closure, Piloerection, Red/crusty deposits, Respiratory rate/character, Salivation), open field observations (Arousal, Backing, Bizarre stereotypic behavior, Convulsions/tremors, Gait, Gait score, Grooming, Mobility, Rearing, Time to first step (seconds), Urination/defecation), sensory observations (Air righting reflex, Approach response, Eye blink response, Forelimb extension, Hind limb extension, Olfactory orientation, Pupil response, Startle response, Tail pinch response, Touch response), neuromuscular observations (Grip strength-hind and forelimb, Hind limb extensor strength, Hind limb foot splay, Rotarod performance), physiological observations (Body temperature, Body weight, Catalepsy)
- Motor activity:
males: during the last week (days 24-28); 5/group
females: lactation day 13; 5/group
Motor activity was measured using the Kinder Scientific Motor Monitor System (Kinder Scientific Company, LLC, Chula Vista, CA). Locomotor activity sessions was performed in a room equipped with a white noise generator set to operate at 70 ± 10 dB.

Oestrous cyclicity (parental animals):

Daily vaginal lavages performed beginning during the pretest period and continuing through the 2-week premating and mating treatment periods until evidence of mating is observed. Lavaging was continued for those females with no evidence of mating until termination of the mating period.

Litter observations:

- Mortality: daily
- Detailed clinical observations: PND 1, 4, 7, 10 and 13. Animals were removed from the cage. 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 Any abnormalities in nesting and nursing behavior were recorded.
- Cage-side observations: daily
- Pup sex: PND 0 or 1, 4 and 13
- Individual pup body weights: PND 1, 4, 7, 10 and 13
- Anogenital distance: PND 1
- Assessment of areolae/nipple retention: PND 13

Postmortem examinations (parental animals):

- Thyroid hormone assessment (T3/T4):
F0: all males at termination and all females on LD 13

- Clinical pathology:
F0 males: on day of scheduled necropsy
F0 females: LD 14

Hematology: Red blood cell count, Hemoglobin concentration, Hematocrit, Mean corpuscular volume, Red blood cell distribution width, Mean corpuscular hemoglobin concentration, Mean corpuscular, hemoglobin, Reticulocyte count (absolute), Platelet count, White blood cell count, Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute), Large unstained cells (absolute), Other cells (as appropriate), Mean platelet volume
Coagulation: Activated partial thromboplastin time, Fibrinogen, Prothrombin time
Clinical chemistry: Alanine aminotransferase, Albumin, Albumin/globulin ratio, Alkaline phosphatase, Aspartate aminotransferase, Bile acids, Calcium, Chloride, Creatinine, Gamma-glutamyltransferase, Globulin (calculated), Glucose, Phosphorus, Potassium, Sodium, Sorbital dehydrogenas, Total bilirubin, Cholesterol, Total protein, Triglycerides, Urea nitrogen

- Necropsy: all F0 animals (all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. For parental females, the number of former implantation sites were recorded).

- Organ weights: all F0 animals; brain, epididymis, pituitary, prostate, seminal vesicle, thyroid, heart, kidney, liver, ovary, spleen, testis, thymus

- Microscopy: all F0 animals (control and high dose group; mid and low dose group in case of observed lesions); aorta, bone marrow (sternum), brain, epidymis, esophagus, eye, adrenal, coagulating gland, pituitary, prostate, salivary gland (mandibular), seminal vesicle, thyroid (incl. parathyroids), heart, kidney, cecum, colon, rectum, liver, lung, lymph nodes (axillary, mandibular, mesenteric), skeletal muscle, macroscopic abnormalities, optic/sciatic nerve, ovary, oviduct, pancreas, peyer's patches, skin, duodenum, ileum, jejunum, spinal cord, spleen, stomach, testis, thymus, trachea, urinary bladder, uterus/cervix, vagina, vas defens

Postmortem examinations (offspring):

- Thyroid hormone assessment (T3/T4):
two pups/litter at PND 4 and one pup/sex/litter at PND 13

- Alizarin Red S staining: in case of suspected skeletal anomlaies
- Gross necopsy: pups older than 5 days (all pups found dead/euthanized in extremis, 1 pup/sex/litter on PND 13)
- preservation of thyroid (incl. parathyroids): 1 pup/sex/litter

Statistics:

All analyses were two-tailed for significance levels of 5% and 1%. All means were presented
with standard deviations.
in-life data:
Continuous data variables (body weights, body weight changes, and food consumption at each interval), estrous cycle length, pre-coital intervals, gestation length, former implantation sites, unaccounted-for sites, thyroid hormone values, anogenital distance (absolute and relative to cube root of body weight), number of nipples/areolae (only when nipples are present), clinical pathology values, and Functional Observational Battery data values will be analyzed by a parametric one-way analysis of variance (ANOVA). If the results of the ANOVA are significant (p<0.05), Dunnett’s test will be applied to the data to compare the treated groups to the control group. Functional Observational Battery parameters which yield scalar or descriptive data will be analyzed by Fisher’s Exact test.
Male copulation, female conception, and male and female mating and fertility indices of the treated groups will be compared to the control group using the Chi-square test with Yates’ correction factor.
Litter data:
The mean litter proportions (% per litter) of pup viability during the postnatal period and sex ratio at birth will be subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup difference. If the results of the ANOVA are significant (p<0.05), Dunn’s test will be applied to the data to compare the treated groups to the control group. Mean litter weights, live litter size, and number of pups born will be subjected to the parametric ANOVA test and Dunnett’s test as described above with the litter representing the experimental unit.
Organ weight data:
Organ weights (absolute and relative to body weights and relative to brain weights) will be subjected to a parametric ANOVA test and Dunnett’s test as described above.
Locomotor activity data:
Two-sided test.

Reproductive indices:

mating index, fertility index, copulation index, female conception index, pre-coital interval

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related clinical observations were noted at the detailed examinations or cage-side observations for males and females at any dietary concentration.

Mortality:

no mortality observed

Description (incidence):

All F0 males and females survived to the scheduled necropsy.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean body weights and body weight gains in the 800, 2500, and 7500 ppm group animals were unaffected by test substance exposure throughout the study.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on hematology parameters in the 800, 2500, and 7500 ppm group males and females when evaluated on Study Day 29 or Lactation Day 14, respectively.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related changes in clinical chemistry were noted for males and females at all dietary concentrations. In the 800, 2500, and 7500 ppm groups, lower mean total bilirubin concentrations were noted for males and females compared to the control group; differences were statistically significant for males at 2500 and 7500 ppm and for females at 7500 ppm.
Additionally, statistically significantly higher mean albumin, total protein, and globulin concentrations were noted for males in the 7500 ppm group compared to the control group. These differences were not considered adverse due to the lack of corresponding adverse microscopic findings.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on thyroid hormone values in the F0 males at any dietary concentration.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Home cage parameters were unaffected by test substance exposure. Handling, open field, neuromuscular and sensory parameters as well as motor activity patterns were unaffected by test substance exposure. There were no statistically significant differences for the test substance-exposure groups when compared to the control group on Study Day 28 (males) or on Lactation Day 13 (females).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Minimal to mild hepatocellular hypertrophy noted in 2500 and 7500 ppm group males and 7500 ppm group females was characterized by increased cytoplasm and increased nuclear diameter. The microscopic distribution of this finding was diffuse in males and centrilobular in females.
Thyroid follicular hypertrophy noted in 2500 and 7500 ppm group males was characterized by increased follicular cell height, increased cytoplasm, and reduced colloid content.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The mean lengths of estrous cycles in these groups were similar to the control group value.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

No test substance-related effects on reproductive performance (mating index, fertility index, copulation index, female conception index, pre-coital interval) were observed at any dietary concentration.

- Gestation Length and Parturition
Mean gestation lengths in the 800, 2500, and 7500 ppm groups were unaffected by test substance exposure. No statistically significant differences were noted. No signs of dystocia were noted in these groups.
- Litter data
The mean number of pups born, live litter size and the percentage of males at birth in 800, 2500, and 7500 ppm groups were similar to the control group values

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

2 500 mg/kg bw/day

Based on:

test mat.

Remarks:

corresponding to 180 and 220 mg/kg bw/d mean substance intake for males and females, respectively

Sex:

male/female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

7 500 ppm

Based on:

test mat.

Remarks:

corresponding to 539 and 668 mg/kg bw/d mean substance intake for males and females, respectively

Sex:

male/female

Basis for effect level:

other: no effects on reproductive performance observed

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

The general physical condition (defined as the occurrence and severity of clinical observations) of all F1 pups in this study was unaffected by test substance exposure.

Mortality / viability:

no mortality observed

Description (incidence and severity):

Postnatal survival in all groups was unaffected by test substance exposure.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean male and female pup body weights and body weight changes during PND 1–13 in the 800, 2500, and 7500 ppm groups were unaffected by parental exposure of the test substance.

Food consumption and compound intake (if feeding study):

not examined

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:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on thyroid hormone values in the F1 males and females at any dietary concentration on PND 13.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distances (absolute and relative to the cube root of pup body weight) in the 800, 2500, and 7500 ppm groups were similar to the control group values

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Areolae/nipple anlagen in the F1 male pups was unaffected by parental exposure to the test substance when evaluated on PND 13. No retained nipples/areolae were noted for any male pup.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No internal findings were noted at the necropsy of pups euthanized on PND 13.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

7 500 ppm

Based on:

test mat.

Remarks:

corresponding to 539 and 668 mg/kg bw/d mean substance intake for males and females, respectively

Sex:

male/female

Basis for effect level:

other: no developmental effects observed

Critical effects observed:

no

Reproductive effects observed:

no

Please refer to attachment

Conclusions:

Based on the lack of effects on F0 reproductive parameters and F1 neonatal survival and growth, a dietary concentration of 7500 ppm (the highest concentration tested) was considered to be the NOAEC for F0 reproductive toxicity, F0 neurotoxicity, and F1 neonatal toxicity. This concentration corresponded to mean dose levels of 539 and 668 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.

Executive summary:

In the present OECD 422 study animals were exposed to the test substance continuously via the diet at concentration of 0, 800, 2500 and 7500 ppm. F0 males were exposed for 14 days prior to mating and continuing until euthanasia for a total of 28 days. F0 females were exposed for 14 days prior to mating and continuing through Lactation Day 13. F1 offspring were potentially exposed to the test substance in utero and via maternal milk, as well as via direct consumption of the diet during the latter portion of the lactation period.

Dietary concentrations of the test substance were adjusted to 0.5x for females that delivered, during Lactation Day 4 through euthanasia, to compensate for the approximately 2x higher maternal food consumption (and consequently higher test substance consumption) that is normally associated with the higher caloric demands of milk production.

The following parameters and end points were evaluated in this study: mortality, clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen retention, neurobehavior, thyroid hormones, clinical pathology (hematology and clinical chemistry), organ weights, and macroscopic and microscopic examinations.

 

Mean calculated test substance consumption:

Males:

58 (800 ppm), 184 (2500 ppm) and 539 (7500 ppm) mg/kg bw/d

Females:

- prior to mating: 52 (800 ppm), 170 (2500 ppm) and 513 (7500 ppm) mg/kg bw/d

- Gestation: 59 (800 ppm), 184 (2500 ppm) and 548 (7500 ppm) mg/kg bw/d

- Lactation days 1 - 4: 94 (800 ppm), 324 (2500 ppm) and 941 (7500 ppm) mg/kg bw/d

- Lactation days 4 - 13: 66 (800 ppm), 200 (2500 ppm) and 669 (7500 ppm) mg/kg bw/d

 

All F0 males and females survived to the scheduled necropsy. No test substance-related clinical observations were noted at the detailed examinations or cage-side observations for males and females at any dietary concentration.

Lower mean food consumption was noted for F0 males and females in the 7500 ppm group during Study Days 0–1 compared to the control group and was considered test substance-related but not adverse due to the transient nature of the changes and based on the lack of corresponding effects on body weight. No other test substance-related effects on body weights, body weight gains, food consumption, and food efficiency were noted for F0 males and females at any dietary concentration throughout the exposure period.

There were no test substance-related effects on FOB parameters (home cage observations, handling observations, open field observations, sensory observations, neuromuscular observations, and physiological observations) or locomotor activity parameters for F0 males when evaluated on Study Day 28 or for F0 females when evaluated on Lactation Day 13 at any dietary concentration.

Higher mean albumin, total protein, and globulin levels were noted in the 7500 ppm group males when compared to the control group. Lower mean total bilirubin levels than the controls were noted in the 800, 2500, and 7500 ppm group males and females; differences were statistically significant in the 2500 ppm group males and the 7500 ppm group males and females. These findings were considered test substance-related but not adverse due to the lack of adverse corresponding microscopic findings. No test substance-related effects on hematology parameters were noted for F0 males and females.

No test substance-related effects on T4 concentrations were noted for F0 males at any dietary concentration.

Exposure by dietary administration resulted in test substance-related, higher liver weights in the 2500 ppm group females and the 7500 ppm group males and females, higher thyroid/parathyroid weights in the 7500 ppm group males, nonadverse hepatocellular hypertrophy and thyroid follicular hypertrophy in the 2500 and 7500 ppm group males, and nonadverse hepatocellular hypertrophy in 7500 ppm group females. The effects on liver weights in the 7500 ppm group males and females were considered adverse due to the magnitude of changes. There were no test substance-related macroscopic observations.

 

The mean number of pups born, live litter size and the percentage of males at birth in 800, 2500, and 7500 ppm groups were similar to the control group values. Postnatal survival in these same groups was unaffected by test substance exposure. The general physical condition (defined as the occurrence and severity of clinical observations) of all F1 pups in this study was unaffected by test substance exposure. Mean male and female pup body weights and body weight changes during PND 1–13 in the 800, 2500, and 7500 ppm groups were unaffected by parental exposure of the test substance. The anogenital distances (absolute and relative to the cube root of pup body weight) in the 800, 2500, and 7500 ppm groups were similar to the control group values. Areolae/nipple anlagen in the F1 male pups was unaffected by parental exposure to the test substance when evaluated on PND 13. No retained nipples/areolae were noted for any male pup. There were no test substance-related effects on thyroid hormone values in the F1 males and females at any dietary concentration on PND 13. No internal findings were noted at the necropsy of pups euthanized on PND 13.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

539 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable without restriction

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD TG 422 and the corresponding dose range finding study performed with 2,4,7,9 -Tetramethyl-5 -decyne-4,7 -diol, ethoxylated (1.3) are available. Animals were exposed to the test substance continuously via the diet at concentration of 0, 800, 2500 and 7500 ppm. F0 males were exposed for 14 days prior to mating and continuing until euthanasia for a total of 28 days. F0 females were exposed for 14 days prior to mating and continuing through Lactation Day 13. F1 offspring were potentially exposed to the test substance in utero and via maternal milk, as well as via direct consumption of the diet during the latter portion of the lactation period. The mean number of pups born, live litter size and the percentage of males at birth in 800, 2500, and 7500 ppm groups were similar to the control group values. Postnatal survival in these same groups was unaffected by test substance exposure. The general physical condition (defined as the occurrence and severity of clinical observations) of all F1 pups in this study was unaffected by test substance exposure. Mean male and female pup body weights and body weight changes during PND 1–13 in the 800, 2500, and 7500 ppm groups were unaffected by parental exposure of the test substance. The anogenital distances (absolute and relative to the cube root of pup body weight) in the 800, 2500, and 7500 ppm groups were similar to the control group values. Areolae/nipple anlagen in the F1 male pups was unaffected by parental exposure to the test substance when evaluated on PND 13. No retained nipples/areolae were noted for any male pup. There were no test substance-related effects on thyroid hormone values in the F1 males and females at any dietary concentration on PND 13. No internal findings were noted at the necropsy of pups euthanized on PND 13.

 

Additionally, a single generation reproduction study was conducted with the structurally related source substances, 2,4,7,9-Tetramethyl-5-decyne-4,7-diol and 2,4,7,9-Tetramethyl-5-decyne-4,7-diol, ethoxylated(3.8). A detailed justification for read-across is attached to iuclid section 13.

 

2,4,7,9-Tetramethyl-5-decyne-4,7-diol was 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 Fo male rats, both test and control, were fed their respective diets until their litters reached the age of 21 days for weaning, when the Fo 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 Fo 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 fertility (95% vs. 100% in control) and lactation (88% vs 98% in control) 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.

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.

 

2,4,7,9-Tetramethyl-5-decyne-4,7-diol, ethoxylated(3.8) was 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. The animals were mated and the newborn raised to weanling age. The weanlings were randomized to their respective groups according to our Standard Operating Procedures and carried on the same levels to the termination of this 91 day feeding study. Body weight and feed consumption data as well as several reproductive parameters were taken from the F0 rats.

Certain hematological and urine analytical parameters were performed on five male and five female F1a rats per group after 45 days and 91 days. Clinical chemistry determinations were conducted on five male and five female F1a rats per group prior to sacrifice. 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 groups while the major organs were examined histopathologically from all the remaining survivors.

The only pertinent findings observed in the F0 parents were the following:

1. Decrease in the weaning weight of the male and female high dose pups,

2.Decrease in the number of live born pups both male and female at the high dose level, Slight decrease in fertility (90% vs. 100% in control) index of the high-dose group,

3.Decreased body weight in all female test groups after weaning.

4.Normal histology of the reproductive organs.

The only pertinent findings observed in the F1a rats were as follows:

1. Only sporadic significant decrease in the feed consumption of the male test groups during the first: eight weeks on test.

2. Biologically significant decrease in the rate of body weight gain of the high dose male and female groups,

3. Normal clinical chemistry values after 91 days on test.

4. Normal hematology findings after 45 and 91 days on test.

5. Normal urinalysis results after 45 and 91 days on test,

6. Dose related increase in the liver to body weight ratios, particularly striking in the female test groups.

From the data it was concluded that, when the test item is fed to rats under the conditions of this experiment, it does not cause any toxic effect at levels of 500 mg/kg/d and 1,000 mg/kg/d. There were no significant effects on the reproductive performance reported.

 

 

Conclusion

Since no effects on reproductive performance and development were derived by an OECD 422 study performed with 2,4,7,9 -Tetramethyl-5 -decyne-4,7 -diol, ethoxylated (1.3), the overall NOAEL used for chemical safety assessment is the value of 539 mg/kg/day.

 

Effects on developmental toxicity

Description of key information

According to OECD 414 the structural analogue, 2,4,7,9-Tetramethyl-5-dcyne-4,7-diol, was administered continuously in the diet during Gestations Days 6 -21.

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.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar toxicological properties because
- they are manufactured from similar precursors under similar conditions
- they share structural similarities with common functional groups: the substances start with an acetylene group as core structure; geminal hydroxyl groups on the alpha carbon atoms; distal to the geminal hydroxyl groups is an isobutyl group (methyl isopropyl); the target substance 2,4,7,9-Tetramethyl-5-decyne-4,7-diol, ethoxylated (1.3) is further functionalised with ethylene oxide and has an ethoxylation degree of 1.3; the source substance 2,4,7,9-Tetramethyl-5-decyne-4,7-diol, ethoxylated (3.8) has an ethoxylation degree of 3.8
- they have similar physicochemical properties and thus, show a similar toxicokinetic behaviour
- they are expected to undergo similar metabolism: oxidation of the terminal methyl groups to result in alcohol, aldehyde and finally the corresponding acid

Therefore, read-across from the existing toxicity, ecotoxicity, environmental fate and physicochemical studies on the source substances is considered as an appropriate adaptation to the standard information requirements of REACH regulation.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see “Justification for read-across” attached to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
see “Justification for read-across” attached to IUCLID section 13

4. DATA MATRIX
see “Justification for read-across” attached to IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Description (incidence and severity):

No remarkable clinical observations were noted at the detailed clinical or cage-side observations at any exposure level.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the scheduled euthanasia on Gestation Day 21.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean maternal body weights, body weight gains, food consumption, food utilization, adjusted body weights, adjusted body weight gains, and gravid uterine weights in the 5000 and 15,000 ppm groups were similar to that in the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

please refer to "body weight and weight changes"

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:

not examined

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):

Remarkable changes in organ weights included dose-responsive higher mean absolute liver weights for females at 5000 and 15,000 ppm; differences from the control group were statistically significant
Differences of mean absolute liver weights in the 5000 and 15000 ppm groups were 27.9 % and 57.9% higher compared to control group values, respectively, higher than the controls. Mean relative liver weights in the 5000 and 15000 ppm groups were 20.4% and 51.0%, respectively, higher than the controls.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

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:

no effects observed

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Remarks:

corresponding to 1126 mg/kg bw/d

Basis for effect level:

organ weights and organ / body weight ratios

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

not examined

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Remarks:

corresponding to 1126 mg/kg bw/d

Sex:

male/female

Basis for effect level:

other:

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

In conclusion, maternal survival, clinical observations, body weight, food consumption, and macroscopic findings and intrauterine growth and survival and external fetal morphology were unaffected at 5000 and 15,000 ppm. Higher mean liver weights were noted for females at 5000 and 15,000 ppm. Based on these results, exposure levels of 1500, 5000, and 15,000 ppm (limit dose) were selected for the subsequent prenatal developmental toxicity study of the test substance offered continuously via the diet to time-mated Crl:CD(SD) rats.

Executive summary:

The objective of this study was to determine dose levels of the source substance, 2,4,7,9-Tetramethyl-5-dcyne-4,7-diol, when administered via the diet, to be evaluated in a definitive prenatal developmental toxicity study in rats.

Animals were exposed continuously to the test substance in the basal diet or the basal diet alone during Gestation Days 6–21.

The following parameters and end points were evaluated in this study: mortality, clinical observations, body weights, body weight gains, gravid uterine weights, food consumption, food efficiency, organ weights, macroscopic examinations, intrauterine growth and survival, and external fetal morphology.

The analyzed dietary formulations contained 99.9% to 109.6% of the test substance which was within the protocol-specified range of target concentrations for suspensions (85% to 115%) and were homogeneous. The test substance was not detected in the analyzed basal diet that was administered to the control group (Group 1). Average test substance compound consumption values were 381 and 1126 mg/kg/day when the entire test substance exposure period is evaluated (GD 6-21), which corresponded to theoretical dietary concentrations of the test substance at 5000 and 15,000 ppm, respectively.

All animals survived to the scheduled euthanasia on Gestation Day 21. All females were gravid with the exception of 1 female in the 5000 ppm group. No remarkable clinical observations were noted at the detailed clinical or cage-side observations at any exposure level.

Mean maternal body weights, body weight gains, food consumption, food utilization, adjusted body weights, adjusted body weight gains, and gravid uterine weights in the 5000 and 15,000 ppm groups were similar to that in the control group.

Remarkable changes in organ weights included dose-responsive higher mean absolute liver weights for females at 5000 and 15,000 ppm; differences from the control group were statistically significant.

Intrauterine growth and survival were unaffected by test substance administration at exposure levels of 5000 and 15,000 ppm. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of live fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight and not statistically significant. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre- implantation loss were similar across all groups. No external developmental variations or malformations were observed in fetuses in this study.

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Based on the comparable chemical structure, physico-chemical, ectoxicological and toxicological properties a testing proposal for a prenatal developmental toxicity study performed with the source substance, 2,4,7,9-Tetramethyl-5-dcyne-4,7-diol and a consequent read across to the target substance 2,4,7,9-Tetramethyl-5-decyne-4,7-diol , ethoxylated was proposed. This approach is also in line with the REACH required animal wellfare considerations. To further strenghten the read across between both Tetramethyl-5-dcyne-4,7-diol derivates two OECD 422 bridging studies with each of them were performed in parallel. Since the results of these studies provide the same mode of action (increased liver weight at the high dose level pointing to an adaptation of enzyme activity as the only significant changes) it was decided to conduct the required developmental toxicity study with the source substance 2,4,7,9-Tetramethyl-5-dcyne-4,7-diol. This tiered testing approach was based on animal wellfare considerations since it was the most effective strategy in reducing animal numbers. However, due to the performance of the two bridging studies as basis for the conclusion on the final testing strategy and for the dose level derivation the study start of the main OECD 414 study is delayed until beginning in January 2022.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar toxicological properties because
- they are manufactured from similar precursors under similar conditions
- they share structural similarities with common functional groups: the substances start with an acetylene group as core structure; geminal hydroxyl groups on the alpha carbon atoms; distal to the geminal hydroxyl groups is an isobutyl group (methyl isopropyl); the target substance 2,4,7,9-Tetramethyl-5-decyne-4,7-diol, ethoxylated (1.3) is further functionalised with ethylene oxide and has an ethoxylation degree of 1.3; the source substance 2,4,7,9-Tetramethyl-5-decyne-4,7-diol, ethoxylated (3.8) has an ethoxylation degree of 3.8
- they have similar physicochemical properties and thus, show a similar toxicokinetic behaviour
- they are expected to undergo similar metabolism: oxidation of the terminal methyl groups to result in alcohol, aldehyde and finally the corresponding acid

Therefore, read-across from the existing toxicity, ecotoxicity, environmental fate and physicochemical studies on the source substances is considered as an appropriate adaptation to the standard information requirements of REACH regulation.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see “Justification for read-across” attached to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
see “Justification for read-across” attached to IUCLID section 13

4. DATA MATRIX
see “Justification for read-across” attached to IUCLID section 13

Reason / purpose for cross-reference:

read-across source

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:

not examined

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 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:

no effects observed

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 effects observed

Remarks on result:

other: detailed data expected to available in Oct 2022

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 the source 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 source substance, 2,4,7,9-Tetramethyl-5-dcyne-4,7-diol, was administered continuously in the diet during Gestations 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

Based on the comparable chemical structure, physico-chemical, ectoxicological and toxicological properties a read across to the results of a prenatal developmental toxicity study performed with the source substance, 2,4,7,9-Tetramethyl-5-dcyne-4,7-diol is performed. This approach is also in line with the REACH required animal wellfare considerations. To further strenghten the read across between both Tetramethyl-5-dcyne-4,7-diol derivates two OECD 422 bridging studies with each of them were performed in parallel. Since the results of these studies provide the same mode of action (increased liver weight at the high dose level pointing to an adaptation of enzyme activity as the only significant changes) it was decided to conduct the required developmental toxicity study with the source substance 2,4,7,9-Tetramethyl-5-dcyne-4,7-diol. This tiered testing approach was based on animal wellfare considerations since it was the most effective strategy in reducing animal numbers. Furthermore, the result of the performed QSAR does not provide any hint for any significant difference in the developmental mode of action between target and source substances.

Additionally, preliminary results of a prenatal developmental dose range finding study performed with the source substance 2,4,7,9-Tetramethyl-5-dcyne-4,7-diol provided no changes in maternal and fetal parameters at doses up to 1000 mg/kg bw/d.

 

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.