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

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 2021 - Jul 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

204-809-1

EC Name:

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

Cas Number:

126-86-3

Molecular formula:

C14 H26 O2

IUPAC Name:

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

Constituent 2

Reference substance name:

2,4,7,9-Tetramethyl-5-dcyne-4,7-diol

IUPAC Name:

2,4,7,9-Tetramethyl-5-dcyne-4,7-diol

Test material form:

other: waxy solid

Details on test material:

Lot Number: 2910-109
Purity: 100 %

Specific details on test material used for the study:

Purity: 99.1 %
Expiry date: 07. Apr. 2025

Test animals

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: approx. 11 weeks
- Weight at study initiation: 269 to 414 g (males)/186 to 255 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: 28 Sept. To: 30. Nov. 2021

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The test material was added to PMI Nutrition International, LLC LabDiet Certified Rodent
Diet 5002 on a weight/weight basis. Diet formulations were prepared at appropriate concentrations to meet dose level requirements.
Frequency of preparation: weekly

Vehicle:

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

Details on oral 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

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.

Duration of treatment / exposure:

Males: for at least 14 days prior to mating and continuing throughout
mating until euthanasia for a total of 29 days.

Females: for at least 14 days prior to mating and continuing throughout
mating, gestation and lactation until euthanasia for a total of 40 or 43 days.

The F1 animals were not directly exposed but were potentially exposed to the test substance in
utero and via milk during lactation.

Frequency of treatment:

continuously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

800 ppm

Remarks:

in diet (corresponding to 56 mg/kg bw/d (males) and 65 mg/kg bw/d (mean 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.

Doses / concentrations 3

Dose / conc.:

2 500 ppm

Remarks:

in diet (corresponding to 174 mg/kg bw/d (males) and 208 mg/kg bw/d (mean 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.

Doses / concentrations 4

Dose / conc.:

7 500 ppm

Remarks:

in diet (corresponding to 518 mg/kg bw/d (males) and 633 mg/kg bw/d (mean 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: 28 d oral toxicity (diet)

Examinations

Observations and examinations performed and frequency:

F0 Generation

- 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 were recorded on the day observed. The observations included, but were 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 (day 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.


F1 Generation

- 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

Sacrifice and pathology:

- Thyroid hormone assessment (T3/T4):
F0: all males at termination and all females on LD 13
F1: two pups/litter at PND 4 and one pup/sex/litter at PND 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; adrenal glands, brain, epididymis, pituitary, prostate, seminal vesicle, thyroid with parathyroids, heart, kidneys, liver, ovaries with oviducts, spleen, testes, 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

F1:
- Alizarin Red S staining: in case of suspected skeletal anomalies
- 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.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related clinical observations were noted in any group
throughout the study.

Mortality:

no mortality observed

Description (incidence):

All F0 males and females in the control, 800, 2500, and 7500 ppm groups survived to the
scheduled necropsies.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean F0 body weights and body weight gains in the 800, 2500, and 7500 ppm group males were
unaffected by exposure to the test substance throughout the study.
Mean body weights and body weight gains in the 800, 2500, and 7500 ppm group females were
unaffected by exposure to the test substance during the premating period, gestation and lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption, evaluated as g/animal/day, in the 800, 2500, and 7500 ppm group
males was comparable to that in the control group during the premating period.
Mean food consumption, evaluated as g/animal/day, in the 800, 2500, and 7500 ppm group
females was comparable to that in the control group during the premating period, gestation. Mean food consumption in the 7500 ppm group was statistically significantly higher than the control group during Lactation Days 4–7 and when the overall lactation period (Days 1–13) were evaluated.

Food efficiency:

not examined

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.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly higher mean albumin, total protein, and globulin levels were noted in
the 7500 ppm group males when compared to the control group. In addition, lower mean total
bilirubin levels were noted in the 800, 2500, and 7500 ppm group males compared to the control
group; the differences were statistically significant. Lower mean alkaline phosphatase, aspartate
aminotransferase, phosphorus and potassium levels, and higher mean cholesterol and sorbitol
dehydrogenase levels were noted in the 7500 ppm group females compared to the control group;
the differences were statistically significant. These findings were considered test
substance-related but not adverse due to the lack of corresponding adverse histopathological
findings. Other statistically significant differences from the control group did not occur in a
dose-related manner and/or the effect occurred in an opposite direction in the other sex,
suggesting no evident relationship to the test substance.

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Home cage, handling, open field, sensory, neuromuscular and physiological parameters, were unaffected by exposure to the test substance.
Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test substance exposure at all concentrations when evaluated on Study Day 28 (males) and Lactation Day 13 (females).

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 (absolute and relative to final body and brain weights) were noted in the 7500 ppm group males and females and correlated with centrilobular hepatocellular hypertrophy noted microscopically. Due to the magnitude of changes (37.0% and 38.5% higher than the controls for F0 males and females, respectively), the effects on liver weight were considered adverse at 7500 ppm in both sexes.
No other test substance-related organ weight changes were noted.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test substance-related internal findings were observed at any exposure level in females that failed to deliver or males and females at the scheduled necropsy.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related centrilobular hepatocellular hypertrophy of the liver was present in the males and females in the 2500 and 7500 ppm groups, and minimal follicular cell hypertrophy of the thyroid glands was noted microscopically in the 800 (females only), 2500, and 7500 ppm group males and females. Hepatocellular hypertrophy correlated with higher mean liver weights in males and females in the 7500 ppm group. The minimal or mild severity, lack of other related microscopic or clinical findings, and frequency of centrilobular and thyroid hypertrophy as responses to xenobiotic exposure support a determination that hypertrophy in both organs was an adaptive, nonadverse finding.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Effect levels

Dose descriptor:

NOAEL

Effect level:

2 500 ppm

Based on:

test mat.

Remarks:

corresponding to 174 and 208 mg/kg bw/d mean substance intake for males and females, respectively

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

serum/plasma biochemistry

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of this screening study, higher mean liver weights in combination with changes in clinical chemistry parameters and hepatocellular hypertrophy were noted at 7500 ppm in both sexes. Therefore, a dietary concentration of 2500 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for F0 systemic toxicity of the test item when administered continuously in the diet to Crl:CD(SD) rats.
This concentration corresponded to mean dose levels of 174 and 208 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 29 days. F0 females were exposed for 14 days prior to mating and continuing through Lactation Day 14. 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:

56 (800 ppm), 174 (2500 ppm) and 518 (7500 ppm) mg/kg bw/d

Females:

- prior to mating: 51 (800 ppm), 166 (2500 ppm) and 482 (7500 ppm) mg/kg bw/d

- Gestation: 56 (800 ppm), 183 (2500 ppm) and 538 (7500 ppm) mg/kg bw/d

- Lactation days 1 - 4: 89 (800 ppm), 291 (2500 ppm) and 910 (7500 ppm) mg/kg bw/d

- Lactation days 4 - 13: 62 (800 ppm), 193 (2500 ppm) and 600 (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.

Mean F0 body weights, body weight gains, and food consumption at 800, 2500, and 7500 ppm were comparable to the control group throughout the study, including during gestation and lactation.

Functional observational battery (FOB) parameters and motor activity at 800, 2500, and 7500 ppm were unaffected by exposure to the test substance.

No test substance-related effects on hematology parameters were noted at any test diet 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 were noted in the 800, 2500, and 7500 ppm group males. Lower mean ALP, AST, phosphorus, and potassium levels, and higher mean cholesterol and SDH levels were noted in the 7500 ppm group 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 mean T4 levels were noted in the F0 males.

Swollen liver was noted in 1 female in the 7500 ppm group at necropsy. Higher mean liver weights (absolute and relative to final body and brain weight) were noted in males and females at the 7500 ppm dose and considered adverse due to the magnitude of changes. Higher liver weights in F0 males and females in the 7500 ppm group correlated microscopically with nonadverse centrilobular hepatocellular hypertrophy in both sexes. Microscopically, minimal to mild centrilobular hepatocellular hypertrophy was present in the 2500 and 7500 ppm groups (males and females), and minimal, nonadverse, thyroid gland follicular cell hypertrophy was also noted in the males and females in the 800 (females only), 2500, and 7500 ppm dose groups.