Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A study with the test item similar to OECD 421 is currently running as a preliminary study to a extended one-generation reproductive toxicity study (basic design) according to OECD guideline 443.


This section will be updated as soon as results are available.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-02-24 to 2021-12-17 (estimated)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Not all parameters measured as the study was conducted as a range finding study for the main OECD 443 study.
GLP compliance:
no
Remarks:
No claim of compliance with Good Laboratory Practice (GLP) will be made for this study; however, the work performed will be followed good scientific practices and adhere to the study plan, any amendments and applicable SOPs.
Limit test:
no
Justification for study design:
The study design was chosen on the basis of OECD guideline 421.
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on species / strain selection:
The Sprague-Dawley rat (sexually mature and virgin) is an accepted species for reproductive toxicity studies by regulatory agencies and historical control data are available.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: (P) 10-12 weeks; (F1) 4 weeks
- Weight at study initiation: (P) Males: 351-400 g; Females: 227-254 g; (F1) Males: will be added when the study is finished; Females: will be added when the study is finished
- Fasting period before study: Not indicated
- Housing: Polycarbonate caging with stainless steel grid (pairing period) or with solid polycarbonate floor (rest of the study) with softwood based bark-free fiber, sterilized by autoclaving
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes: Air will be filtered, not recirculated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 0 to approx. day 60 (F0 females), from day 0 to approx. day 37 (F0 males), from approx. day 37 to approx. day 91 (F1 offspring)
Route of administration:
oral: feed
Vehicle:
other: Will be documented in the study data and included in the final report.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Unless advised otherwise, the procedure for production of a premix includes grinding the test item/diet admixture.

DIET PREPARATION
- Rate of preparation of diet: Weekly, and up to one week in advance of first feeding.
- Mixing appropriate amounts with: SDS VRF1
- Storage temperature of food: Prepared diets will be stored frozen (-10 to -30 °C). Formulated diets may be stored at ambient temperature (15 to 25ºC) (once defrosted), within the confirmed stability period of up to 8 days, pending feeding and accounting for the time in the food hoppers.
Details on mating procedure:
- M/F ratio per cage: 1.1
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy.
- Further matings after two unsuccessful attempts: A female showing no evidence of mating after completion of the pairing period will be separated from the male and vaginal smearing will be continued for up to five days or until the first estrus smear is seen. If an estrus smear is seen during this period, the female cannot be pregnant and will be dispatched to necropsy as soon as practically possible. If an estrus smear is not seen the female will be dispatched to necropsy on or after day 25 after the last day of pairing.
- After successful mating each pregnant female was caged: Singly to permit collection of food consumption data individually for pregnant females and during lactation.
Analytical verification of doses or concentrations:
no
Remarks:
No formulation analysis will be performed on this study but was tested as part of another study (see below).
Details on analytical verification of doses or concentrations:
The homogeneity and stability of the test item in the diet during storage were confirmed as part of another study. In that study, prepared diets in the range 1500 to 15000 ppm were determined to be stable for:
- 8 days at ambient temperature (15 to 25 °C)
- 15 days when stored frozen (-10 to -30 °C)
Prepared diets will be stored frozen (-10 to -30 °C) until required for feeding.
Duration of treatment / exposure:
F0 females: Two weeks before pairing until Day 21 of lactation (approx. 60 days); F0 males: two weeks before pairing until after successful littering by females (approx. 37 days); F1 litters: From late lactation when offspring start to consume diet (approx. in the age of 28 days) to approximately week 7 of age after sexual maturity has been attained (approx. 26 days)
Frequency of treatment:
Continuously
Dose / conc.:
0 ppm
Remarks:
control
Dose / conc.:
3 750 ppm (nominal)
Remarks:
Expected achieved doses: Approx. 225 mg/kg/day (males) and 235 mg/kg/day (females)
Dose / conc.:
7 500 ppm (nominal)
Remarks:
Expected achieved doses: Approx. 456 mg/kg/day (males) and 485 mg/kg/day (females)
Dose / conc.:
15 000 ppm (nominal)
Remarks:
Expected achieved doses: Approx. 886 mg/kg/day (males) and 842 mg/kg/day (females)
No. of animals per sex per dose:
8 animals per sex per dose (F0 generation), 10 animals per sex per dose (F1 generation)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dietary levels for this study have been selected in consultation with the sponsor, based on the results of a 21-Day palatability study in Sprague Dawley Rats by dietary administration (see section 7.5.1). In this study, no premature decedents or treatment-related changes in clinical conditions were observed and only some slight effects on body weight and liver weight were observed. Thus the highest dose used in the palatibility study (15000 ppm, resulting in mean achieved doses of 886 mg/kg/day in males and 842 mg/kg/day in females) was chosen also for this dose-range finding test.
- Fasting period before blood sampling for clinical biochemistry: Animals were not fasted before blood sampling and clinical biochemistry.
Positive control:
Not included
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations included: Evidence of reaction to treatment or ill-health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice weekly; days 0, 3, 7, 10, 14, 18 and 20 after mating and days 1, 4, 7, 10, 14, 18 and 21 of lactation for F0 females.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly and at the day of necropsy (F0 males), twice weekly until mating detected; days 0, 3, 7, 10, 14, 17 and 20 after mating and on days 1, 4, 7, 11, 14, 18 and 21 of lactation (F0 females); days 21, 23, 25, 27 and 28 of age and twice weekly from nominal 4 weeks of age to termination at approximately 7 weeks of age (F1 animals)

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

HEMATOLOGICAL ANALYSIS
- Time schedule for examinations: At termination (no overnight deprivation of food)
- Anaesthetic: Isoflurane
- Parameters examined: Thyroid hormones, hematocrit, hemoglobin concentration, erythrocyte count, total leucocyte count, differential leucocyte count, platelet count, mean cell hemoglobin, mean cell volume, mean cell hemoglobin concentration, red cell distribution width, prothrombin time, activated partial thromboplastin time
Oestrous cyclicity (parental animals):
The oestrus cycle will be determined by dry smears (for 15 days before pairing, using cotton swabs) and by wet smears (after pairing until evidence of mating confirmed, using pipette lavage). Percentages of females showing the following cycle types will be calculated:
- Regular (all observed cycles are of 4 or 5 days; may be divided into cycles of 4, 4 and 5, and 5 days, if inter-group differences are apparent),
- Irregular: At least one cycle of 2, 3 or 6 to 10 days.
- Acyclic: At least 10 days without estrus (beginning before pairing).
Sperm parameters (parental animals):
Sperm parameters will not be examined.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- A maximum of 5 pups/sex/litter were selected. Excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Number and sex of pups, sex ratio, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical and behaviour abnormalities, anogenital distance (AGD), gross evaluation of external genitalia

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities with assessment of stomach for presence of milk, where possible. Abnormal tissues retained in an appropriate fixative.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, after successful littering by females
- Maternal animals: All surviving animals on day 21 of lactation. If females will fail to produce viable litter on day 25 after mating, if litters die before weaning, on day last offspring dies. If females fail to mate, on or after day 25 after last day of pairing OR day of confirmed estrus after failure to mate.

GROSS NECROPSY
- Gross necropsy consists of external examinations (see table 1).

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in table 1 will be prepared for microscopic examination and weighed, respectively. Any abnormal tissues retained and may be weighed at the discretion of necropsy staff. In addition, the following will be recorded for all F0 females (including those prematurely sacrificed, where possible): number of implantation sites, mammary tissue appearance for females whose litter dies before day 21 of lactation.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected are sacrificed at 4 days of age.
- These animals are subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Culled offspring with clinical signs on day 4 of age will be subject to complete macroscopic examination with assessment of stomach for presence of milk, where possible. Abnormal tissues retained in an appropriate fixative. Culled offspring with no clinical sign on Day 4 of age will be killed and discarded without necropsy examination.

GROSS NECROPSY
- Gross necropsy consisted of complete external examinations. Organs are weighted (see table 2).

HISTOPATHOLOGY / ORGAN WEIGTHS
Abnormal tissues will be retained in an appropriate fixative and checked histopathologically (see table 2).
Statistics:
The following data types will be analyzed at each timepoint separately, where required, in support of interpretation: body weight, using absolute weights and gains over appropriate study periods; food consumption, over appropriate study periods; organ weights, both absolute and relative to body weight estrous cycles and vaginal opening to first estrous; pre-coital interval; mating performance and fertility; gestation length and gestation index; litter data; thyroid hormones.

For categorical data, the proportion of animals will be analyzed for each treated group (as appropriate) versus the control group. For continuous data, Bartlett’s test will first be applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett’s test, treated groups will then be compared with the control group, incorporating adjustment for multiple comparisons where necessary.
Reproductive indices:
Percentage mating (Number animals mating x 100 / Animals paired), conception rate (Number animals achieving pregnancy x 100 / Animals mated), fertility index (number animals achieving pregnancy x 100 / Animals paired)
Offspring viability indices:
Post-implantation survival index (Total number offspring born x 100 / Total number uterine implantation sites), Live birth index (Number live offspring on Day 1 after littering x 100 / Total number of offspring born), Viability index (Number live off spring on Day 4 before culling x 100 / Number live off spring on Day 1 after littering), Lactation index (Number live off spring on Day 21 after littering x 100 / Number live off spring on Day 4 (after culling)
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence):
Will be updated after completion of the study.
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Will be updated after completion of the study.
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Will be updated after completion of the study.
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Will be updated after completion of the study.
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Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Key result
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Will be updated after completion of the study.
Key result
Critical effects observed:
not specified
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
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Will be updated after completion of the study.
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Will be updated after completion of the study.
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Will be updated after completion of the study.
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Will be updated after completion of the study.
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Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
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Will be updated after completion of the study.
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Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Will be updated after completion of the study.
Key result
Critical effects observed:
not specified
Key result
Reproductive effects observed:
not specified
Executive summary:

An oral feeding study with the test item in male and female Sprague Dawley rats similar to OECD 421 is currently running as a preliminary study to a extended one-generation reproductive toxicity study (basic design) according to OECD guideline 443. The nominal dose range was chosen between  0 and 15000 ppm, which is expected to result in actual achieved doses of approx. 225 to 886 mg/kg bw/day (as determined in a 21-day palatability study, please refer to section 7.5.1). Clinical signs, body weight, food consumption, hematology, gross pathology estrus cyclicity and number of implantation sites + mammary tissue appearance (for females whose litter die before day 21 of lactation) will be examined in the P0 generation. Litters will be evaluated for number and sex of pups, sex ratio, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical and behaviour abnormalities, anogenital distance (AGD) and gross evaluation of external genitalia. Reproductive indices and offspring viability indices will be calculated. This section will be updated with the results of the EOGRS soon as the study is finalized. 

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to reproduction, screening for reproductive/developmental toxicity, RL2


An oral feeding study with the test item in male and female Sprague Dawley rats similar to OECD 421 is currently running as a preliminary study to a extended one-generation reproductive toxicity study (basic design) according to OECD guideline 443. The nominal dose range was chosen between  0 and 15000 ppm, which is expected to result in actual achieved doses of approx. 225 to 886 mg/kg bw/day (as determined in a 21-day palatability study, please refer to section 7.5.1). Clinical signs, body weight, food consumption, hematology, gross pathology estrus cyclicity and number of implantation sites + mammary tissue appearance (for females whose litter die before day 21 of lactation) will be examined in the P0 generation. Litters will be evaluated for number and sex of pups, sex ratio, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical and behaviour abnormalities, anogenital distance (AGD) and gross evaluation of external genitalia. Reproductive indices and offspring viability indices will be calculated. This section will be updated with the results of the EOGRS soon as the study is finalized. 


 


Toxicity to reproduction, extended one-generation reproductive toxicity study


An extended one-generation reproductive toxicity study has not been conducted with the test item. In a 90-day repeated dose oral gavage study in rats with the analogue chemical, dimyrcetol, there were no effects noted in female rats on reproductive organs or oestrous cyclicity at the highest dose level tested (1000 mg/kg bw/day). In male rats, spermatid counts in the testis at the highest dose were significantly reduced versus the control animals at the highest dose level. There were no similar reductions in spermatid counts in the cauda epididymis and no histopathological changes were associated with the reduced sperm counts. No further conclusions can be drawn from these findings.


 


According to Annex IX of Regulation (EC) No 1907/2006, it is laid down that a one-generation reproductive toxicity test shall be performed by the most appropriate route of administration and having regard to the likely route of human exposure. Given the low vapor pressure of the test material, exposure by the inhalation route is expected to be minimal. Dermal exposure in humans is anticipated, but in vitro dermal absorption testing with human skin of the analogue material, dimyrcetol, indicates only minimal potential dermal absorption (see Section 7.1.2).


 


Based on an ECHA decision (decision number TPE-D-2114449866-32-01/F), an extended one-generation reproductive toxicity study (test method OECD TG 443) in rats by the oral route has been requested. The test substance is currently tested in a dose-range finding test similar to OECD TG 421 (see section 7.8.1).  After this study is completed, the requested OECD TG 443 study will be started. The test design will follow the subsequent study-design specifications made by ECHA:


 



  • Ten weeks premating exposure duration for the parental (P0) generation;

  • Dose level setting shall aim to induce systemic toxicity at the highest dose level;

  • Cohort 1A (Reproductive toxicity);

  • Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation.


 


Sufficient mating pairs to produce 20 animals per dose group (P generation) will be used. Cohort 1B will not be extended to produce the F2 generation, as the substance is not classified as mutagenic in vivo (cat. 2), there is no evidence that the internal dose of the substance/metabolites reaches an equilibrium state only after prolonged exposure, and there is no evidence from in vivo studies or other methods that there are relevant modes of action related to endocrine disruption. No inclusion of cohorts 2A and 2B (developmental neurotoxicity) and cohort 3 (developmental immunotoxicity) is intended as no signs of neurotoxicity or immunotoxicity of the substance has been observed in any of the repeated dose toxicity studies. A ten-week premating exposure duration for the parental (P0) generation is proposed. According to the ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7a: Endpoint specific guidance (version 6.0), the starting point for deciding on the length of premating exposure period should be ten weeks to cover the full spermatogenesis and folliculogenesis before mating, allowing meaningful assessment of the effects on fertility. Ten weeks premating exposure duration is required because there is no substance specific information in the dossier supporting shorter premating exposure duration. Reduced spermatid counts at the highest dose level (1000 mg/kg bw/day) with no similar reductions in the cauda epididymis were noted in the testes of male rats in a subchronic oral toxicity study according to OECD guideline 408 with the analogue substance dimyrcetol. Because of some methodological and interpretational inconsistencies, and because only the highest dose level was assayed for spermatid counts in this study, no further conclusions were drawn concerning the significance of these findings. Thus, in the planned study, the total number of homogenization-resistant testicular and cauda epididymal spermatid counts shall be enumerated. An extension of the study is not necessary as potential effects on spermatids can be identified in the P0 generation and an extension of the study to a second generation would not provide any additional information. Dose level selection will be based on the dose range finding study currently running. A dose which induces some signs of toxicity will be selected as highest dose for the OECD TG 443 study.


 


This section will be updated with the results of the EOGRS soon as the study is finalized. 


 


Please refer to Section 13 of the file for read-across documentation and rationale for the use of analogue chemicals for dihydromyrcenol.

Effects on developmental toxicity

Description of key information

On the basis of the data from a developmental toxicity study in rats, meeting the requirements of the Food and Drug Administration for the evaluation of ICH Harmonized Tripartite Guideline stages C and D of the reproductive process with the analogue substance dimyrcetol, the maternal no-observable-adverse-effect-level (NOAEL) was 1000 mg/kg bw/day. The 500 mg/kg bwt/day dose level was assigned as the NOEL for maternal effects. The developmental NOAEL was also 1000 mg/kg bw/day. The 500 mg/kg bw/day dose level was assigned as the NOEL for developmental effects.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
other: US FDA; This study was designed to evaluate ICH Harmonised Tripartite Guideline stages C and D of the reproductive process
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Cr1:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at arrival: 62 days (approximate)
- Weight at study initiation: 218 to 244 g
- Fasting period before study: None
- Housing: Individually in stainless steel, wire-bottomed cages (except during cohabitation)
- Diet: Ad libitum. Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, MO)
- Water: Ad libitum. Local water processed by reverse osmosis. Chlorine added as bacteriostat
- Acclimation period: Yes, but duration not stated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8-26.1 (targeted)
- Humidity (%): 30 to 70 (targeted)
- Air changes (per hr): 10 (minimum)
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test article were prepared weekly at the testing facility. Prepared formulations were stored at room temperature, protected from light.

VEHICLE
- Concentration in vehicle: The dosage preparations are shown in Table 1.
- Amount of vehicle: Constant dosage volume of 10 mL/kg
Details on analytical verification of doses or concentrations:
Samples were analyzed according to the method described in Charles River Laboratories Preclinical Services. Results of concentration and homogeneity analyses were within +/- 15% of calculated concentrations and
Details on mating procedure:
After a suitable acclimation, 130 virgin female rats were placed into cohabitation with 130 breeder male rats, one male rat per female rat. The cohabitation period was a maximum of five days. Gestation day 0 was considered the first day when spermatozoa were detected in a vaginal smear or a copulatory plug was observed. Pregnant rats were assigned to individual housing.
Duration of treatment / exposure:
Rats were treated on gestation days 7 through 17. Dosages were adjusted daily based on the individual body weights recorded prior to dosage administration.
Frequency of treatment:
Once daily
Duration of test:
Until gestation day 21
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle
No. of animals per sex per dose:
25 female/dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages were based on the results of a range-finding study with the same test article at a dosage level of 1000 mg/kg/day. This was selected as the highest dose in the current study.
- Rationale for route of administration: Oral (gavage) was selected for use as comparison with the dietary route; the exact dosage can be accurately determined.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (rats were observed for viability at least twice daily each day of the study and weekly for clinical observations and general appearance during the acclimation period and on DG 0)
- Cage side observations included: Clinical observations, abortions, premature deliveries and deaths before and approximately 3 hours after dosing, and once daily during the post-dosage period.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during the acclimation period, on DG 0 and daily during the dosage and post-dosage periods

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: Thoracic, abdominal and pelvic viscera. Gross lessions were retained in neutral-buffered 10% formalin for possible future evaluation. Unless specifically cited, all other tissues were discarded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Placentae were examined for size, color and shape. Number and distribution of live and dead fetuses were recorded.
Fetal examinations:
- External examinations: Yes (all per litter)
- Soft tissue examinations: Yes (half per litter)
- Skeletal examinations: Yes (half per litter)
- Head examinations: No data
- Anogenital distance of all live rodent pups: Not specified
Statistics:
Clinical observations and other proportional data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution. Continuous data (e.g., body weights, body weight changes, feed consumption values, and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights and fetal anomaly data) were analyzed using Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e., Bartlett’s Test was not significant (p>0.001)]. If the Analysis of Variance was significant (p≤0.05), Dunnett’s Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett’s Test was significant (p≤0.001)], the Kruskal-Wallis Test was used, when less than or equal to 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p≤0.05), Dunn’s Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher’s Exact Test was used to analyze the data. Count data obtained at Caesarean-sectioning of the dams were evaluated using the procedures described above for the Kruskal-Wallis Test.
Historical control data:
Historical control data was available from this laboratory for the period from January 2005 to January 2007.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Minor incidences of sparse hair coat or localized alopecia, excess salivation, urine-stained abdominal fur, ungroomed coat, rales, ptosis, chromodacryorrhea (colored tears), and soft or liquid feces. These observations were all considered unrelated to the treatment because the incidences were observed in vehicle control rats, were not dosage dependent, or were transient and did not persist.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: Body weight gains for the entire dosage period were reduced by 5% when compared with controls, an observation that was not statistically significant but was considered evidence of a threshold for maternal toxicity.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: Absolute (g/d) and relative (g/kg/d) feed consumption values were significantly reduced for the entire dosage period.



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
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross lesions were noted at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
not examined
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
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: Body weights for combined male and female fetuses were reduced approximately 3%. The reduction in female fetuses was statistically significant (P ≤ .05).
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
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Significant increases in fetal variations attributable to the test article were observed only at the 1000 mg/kg bw/day dosage level. These changes consisted of reversible minor variations, including a threshold but statistically significant increase in supernumerary ribs, along with associated significant increases and decreases in the respective numbers of thoracic and lumbar vertebrae, and a small but statistically significant retardation in ossification of the metatarsal bones in the hindpaws, evident as a reduction in the mean number of ossified metatarsal bones.
Key result
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
not specified

Table 2: MATERNAL BODY WEIGHT GAINS - SUMMARY






















































































































































DOSE GROUP



 



I



II



III



IV



DOSAGE (mg/kg bwt/day)



 



0 (control)



250



500



1000



RATS TESTED



N



25



25



25



25



PREGNANT



N



22



24



24



25



MATERNAL BODY



 



 



 



 



 



WEIGHT CHANGE (G)



 



 



 



 



 



DAYS 0 - 7



MEAN±S.D.



+42.5 ± 7.2



+42.0 ± 8.2



+40.7 ± 10.6



+39.8 ± 7.5



DAYS 7 - 8



MEAN±S.D.



-2.6 ± 4.6



-0.4 ± 5.2



-1.2 ± 5.5



-4.1 ± 6.8



DAYS 8 - 9



MEAN±S.D.



+1.4 ± 4.3



+2.0 ± 5.6



+1.5 ± 4.0



-1.1 ± 6.2



DAYS 9 - 10



MEAN±S.D.



+3.2 ± 4.6



+4.0 ± 4.8



+6.1 ± 4.2



+5.4 ± 5.7



DAYS 7 - 10



MEAN±S.D.



+2.0 ± 6.5



+5.5 ± 7.0



+6.4 ± 8.6



+0.1 ± 9.4



DAYS 10 - 12



MEAN±S.D.



+12.0 ± 5.3



+14.8 ± 5.4



+12.9 ± 5.2



+11.8 ± 9.6



DAYS 12 - 15



MEAN±S.D.



+17.7 ± 8.5



+17.1 ± 9.5



+19.8 ± 5.8



+18.4 ± 7.3



DAYS 15 - 18



MEAN±S.D.



+41.2 ± 9.6



+39.4 ± 8.8



+41.2 ± 7.3



+39.0 ± 7.7



DAYS 7 - 18



MEAN±S.D.



+73.0 ± 17.0



+76.8 ± 15.2



+80.2 ± 14.5



+69.3 ± 15.1



DAYS 18 - 21



MEAN±S.D.



+59.4 ± 11.4



+54.2 ± 8.7



+61.0 ± 10.8



+58.1 ± 12.8



DAYS 7 - 21



MEAN±S.D.



+132.4 ± 22.6



+130.9 ± 16.7



+141.2 ± 20.5



+127.4 ± 20.5



DAYS 0 - 21



MEAN±S.D.



+174.9 ± 25.6



+173.0 ± 21.0



+181.9 ± 27.2



+167.2 ± 23.6



 


 


Table 3: MATERNAL RELATIVE FEED CONSUMPTION VALUES (G/KG/DAY) - SUMMARY





































































































































































DOSAGE GROUP



I



II



III



IV



 



DOSAGE (MG/KG/DAY)a



 



0 (VEHICLE)



250



500



1000



RATS TESTED



N



25



25



25



25



PREGNANT



N



22



24



24



25



MATERNAL FEED



 



 



 



 



 



CONSUMPTION (G/KG/DAY)



 



 



 



 



DAYS 0 - 7



MEAN±S.D.



96.2 ± 6.8



94.2 ± 6.9



93.5 ± 10.6



92.5 ± 6.5



 



 



 



[ 23]b



[ 23]b



 



DAYS 7 - 10



MEAN±S.D.



58.6 ± 10.2



60.8 ± 9.7



60.4 ± 12.7



46.2 ± 11.3**



 



 



 



 



[ 23]b



 



DAYS 10 - 12



MEAN±S.D.



61.4 ± 9.6



65.4 ± 8.8



60.0 ± 7.9



54.2 ± 15.5*



DAYS 12 - 15



MEAN±S.D.



59.8 ± 7.2



63.5 ± 9.7



59.6 ± 7.1



54.9 ± 9.3



 



 



 



[ 22]b



[ 23]b



 



DAYS 15 - 18



MEAN±S.D.



63.8 ± 14.0



68.7 ± 7.6



64.2 ± 10.0



60.8 ± 8.9



 



 



[ 21]b



 



 



 



DAYS 7 - 18



MEAN±S.D.



61.0 ± 5.6



64.5 ± 6.0



61.4 ± 7.3



54.1 ± 6.1**



 



 



[ 21]b



 



 



 



DAYS 18 - 21



MEAN±S.D.



73.1 ± 8.1



71.8 ± 8.7



74.6 ± 7.1



76.2 ± 8.1



DAYS 7 - 21



MEAN±S.D.



63.7 ± 4.8



66.4 ± 5.4



64.7 ± 6.0



59.6 ± 5.3*



DAYS 0 - 21



MEAN±S.D.



69.5 ± 4.0



70.5 ± 4.6



69.0 ± 5.3



66.0 ± 3.8*



[ ] = NUMBER OF VALUES AVERAGED



  1. Dosage occurred on days 7 through 17 of gestation.

  2. Excludes values that could not be calculated or appeared incorrectly recorded, as well as those associated with spillage.


* Significantly different from the vehicle control group value (p≤0.05). ** Significantly different from the vehicle control group value (p≤0.01).


 


 


Table 4: Dosage-Dependent Statistically Significant Fetal Observations (Average Ossification Sites per Fetus per Litter)


 




























































































































































































Observation



Dosage Group (DGs 7 through 17)



 



 



mg/kg/day



0 (Vehicle)



250



500



1000



Historical Controla



 



 



 



 



 



 



Litters



22



24



24



25



982



Fetuses



155



164



183



184



7305



Rats tested, n



25



25



25



25



 



Rats pregnant, n (%)



22 (88)



24 (96)



24 (96)



25 (100)



 



 



 



 



 



 



 



Corpora lutea, mean ± SD



14.6 ± 2.4



14.9 ± 2.8



16.3 ± 2.4



15.8 ± 2.3



 



Implantations, mean ± SD



14.0 ± 2.1



13.8 ± 2.8



15.2 ± 1.7



14.8 ± 1.8



 



Resorptions, mean ± SD



0.6 ± 0.7



0.8 ± 0.9



0.6 ± 0.6



0.5 ± 1.2



 



Litter size, mean ± SD



13.4 ± 2.2



13.0 ± 2.4



14.6 ± 2.0



14.3 ± 2.4



 



Fetal body weight/litter, mean ± SD



5.50 ± 0.28



5.54 ± 0.30



5.55 ± 0.31



5.33 ± 0.33



 



Male fetuses



5.63 ± 0.28



5.73 ± 0.31



5.68 ± 0.28



5.45 ± 0.36



 



Female fetuses



5.38 ± 0.29



5.37 ± 0.28



5.42 ± 0.35



5.18 ± 0.32*



 



Live fetuses/group



295



311



350



358



 



Dead fetuses/group



0



0



0



0



 



 



 



 



 



 



 



Ribs (pairs)b,c



13.06 ± 0.11



13.06 ± 0.10



13.09 ± 0.13



13.19 ± 0.23d



13.06 (13.02-13.12)



Thoracic Vertebraeb,c



13.08 ± 0.15



13.08 ± 0.16



13.12 ± 0.17



13.24 ± 0.28*,d



13.08 (13.02-13.16)



Lumbar Vertebraeb,c



5.92 ± 0.15



5.91 ± 0.16



5.87 ± 0.17



5.75 ± 0.27**,d



5.92 (5.83-5.97)



Metatarsalsb,c



4.95 ± 0.12



4.87 ± 0.21



4.91 ± 0.12



4.78 ± 0.27**,d



4.83 (4.68-4.95)



 



 



 



 



 



 




  1. Studies (N = 44) conducted at the Testing Facility between January 2005 and January 2007 in rats of the same strain.

  2. Means and Standard Deviations

  3. Mean and range of means per study

  4. Bold = effect of test article because values were significant and/or exceeded historical ranges. DGs = Days of Gestation


* = Significantly different from the vehicle control group value (p≤0.05). ** = Significantly different from the vehicle control group value (p≤0.01).


Testing facility historical control data for mean fetal body weight ranges (82 studies): combined male/female, 4.94 to 5.93 g; male fetuses, 5.10 to 5.98 g; female fetuses, 4.80 to 5.78 g.


 

Conclusions:
On the basis of these data, the maternal no-observable-adverse-effect-level (NOAEL) was 1000 mg/kg bw/day. At this dose level, maternal body weight gains were reduced (not significantly) and feed consumption was significantly reduced. The 500 mg/kg bwt/day dose level was assigned as the NOEL for maternal effects.

The developmental NOAEL was also 1000 mg/kg bw/day. This was based on minimal reductions in female fetal weights and small increases in reversible variations in skeletal ossification. The 500 mg/kg bw/day dose level was assigned as the NOEL for developmental effects.
Executive summary:

The test item was administered in corn oil by gavage to groups of pregnant Sprague-Dawley rats at dosages of 0, 250, 500 or 1000 mg/kg bw/day on gestational days 7 to 17. Viability, clinical signs, body weights and feed consumption were observed or measured for all animals. Animals were sacrificed on gestational day 21 and Caesarean sectioning performed. Fetuses were weighed and examined for sex, gross changes, and soft tissue or skeletal alterations. No clinical signs attributable to the test item administration were observed. At the 1000 mg/kg bw/day dose level, mean maternal body weight gains were reduced 5% versus controls. Absolute and relative feed consumption were also reduced at this dose level. The minimal maternal effects noted were associated with minimal (approx. 3%) reductions in fetal body weights, reversible variations in ossification and increases in supernumerary thoracic ribs with associated increases or decreases in thoracic and lumbar vertebrae, respectively. Findings of delayed skeletal ossification are generally considered readily reversible (Carney and Kimmel, 2007 "Interpretation of skeletal variations for human risk assessment: Delayed ossification and wavy ribs", Birth Defects Research 80:473 -496). The minimal developmental delays (reduced pup weights, delayed ossificaiton, changes in metatarsals) are all expected to be readily reversible based on studies that have investigated the progression and outcome of similar effects. On the basis of these data, the maternal no-observable-adverse-effect-level (NOAEL) was 1000 mg/kg bw/day. At this dose level, maternal body weight gains were reduced (not significantly) and feed consumption was significantly reduced.  The 500 mg/kg bwt/day dose level was assigned as the NOEL for maternal effects.The developmental NOAEL was also 1000 mg/kg bw/day.  This was based on minimal reductions in female fetal weights and small increases in reversible variations in skeletal ossification. The 500 mg/kg bw/day dose level was assigned as the NOEL for developmental effects.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Guideline study with acceptable restrictions
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

Developmental toxicity, rat, RL2


There is no information available on the developmental toxicity of dihydromyrcenol.


 


Developmental toxicity was determined in rats following the GLP and the ICH Harmonized Tripartite Guidelines (Stages C and D of the reproductive process) with the analogue chemical, dimyrcetol. The test item was administered in corn oil by gavage to groups of pregnant Sprague-Dawley rats at dosages of 0, 250, 500 or 1000 mg/kg bw/day on gestational days 7 to 17. Viability, clinical signs, body weights and feed consumption were observed or measured for all animals. Animals were sacrificed on gestational day 21 and Caesarean sectioning performed. Fetuses were weighed and examined for sex, gross changes, and soft tissue or skeletal alterations. No clinical signs attributable to the test item administration were observed. At the 1000 mg/kg bw/day dose level, mean maternal body weight gains were reduced 5% versus controls. Absolute and relative feed consumption were also reduced at this dose level. The minimal maternal effects noted were associated with minimal (approx. 3%) reductions in fetal body weights, reversible variations in ossification and increases in supernumerary thoracic ribs with associated increases or decreases in thoracic and lumbar vertebrae, respectively. Findings of delayed skeletal ossification are generally considered readily reversible (Carney and Kimmel, 2007 "Interpretation of skeletal variations for human risk assessment: Delayed ossification and wavy ribs", Birth Defects Research 80:473 -496). The minimal developmental delays (reduced pup weights, delayed ossificaiton, changes in metatarsals) are all expected to be readily reversible based on studies that have investigated the progression and outcome of similar effects. On the basis of these data, the maternal no-observable-adverse-effect-level (NOAEL) was 1000 mg/kg bw/day. At this dose level, maternal body weight gains were reduced (not significantly) and feed consumption was significantly reduced.  The 500 mg/kg bwt/day dose level was assigned as the NOEL for maternal effects.The developmental NOAEL was also 1000 mg/kg bw/day.  This was based on minimal reductions in female fetal weights and small increases in reversible variations in skeletal ossification. The 500 mg/kg bw/day dose level was assigned as the NOEL for developmental effects.


 


Developmental toxicity, rabbit


A second study on developmental toxicity in a second species is legally required according to REACH Annex X. Furthermore, none of the adaption options from Annex X, 8.7, column 2 are applicable for the substance. Thus, based on the legal requirements, a Prenatal Developmental Toxicity Study on a second species according to OECD guideline 414 is proposed to assess the effects of the test substance on growth, survival, maintenance of pregnancy of maternal animals and effects on resorptions, fetal deaths fetal growth, morphological variations and malformations of the offsprings of a species other than the rat. This study is the test system proposed in Annex X, 8.7, column 1 and in the ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R 7a: Endpoint specific guidance, 2017. Under consideration of the substance properties, it is regarded to be the appropriate test system to investigate the potential to induce developmental toxicity, i. e. substances or their metabolites (hydrolysis products) that become readily systemically available upon ingestion. Furthermore, historical control data are available for various tissues. The test substance is proposed to be administered orally by gavage to rabbits as the proposed non-rodent species according to OECD guideline 414. As no repeated dose toxicity data are available in rabbits, doses will be based on a dose range finding test which will be conducted prior to the main experiment. It is noted that the additional time period required for the dose range finding test should also be considered in setting the deadline for submission of data. Based on the results of the dose range finding test and taken the OECD requirements into consideration, a maximum dose will be chosen as a maximum tolerated dose or, if no effects occur, as the maximum recommended dose of 1000 mg/kg bw/day. Two additional doses (separated by a factor of 2 to 4) are proposed. An appropriate vehicle will be selected in a solubility study together with the testing laboratory, also taking into account the compatibility with the species. Details will be defined in the study protocol. A sufficient number of female rabbits per dose or control groups to reach a minimum of 20 animals with implantation sites at necropsy will be chosen and is proposed. The test chemical is intended to be administered daily to pregnant animals by gavage, at least from implantation to one day prior to the day of scheduled killing. Shortly before caesarean section, the females will be killed, the uterine contents are examined, and the fetuses are evaluated for fetal deaths, sex, body weight, AGD, soft tissue and skeletal changes. Dams will be observed during the treatment period, weighted, and examined post-mortem. These examinations will include macroscopical structural abnormalities, weight of thyroid glands and uteri, histopathological assessment of thyroid gland and uteri, number of corpora lutea and degree of resorption.


 


For the full study proposal, please refer to section 7.8.2. This section will be updated as soon as experimental data are available.


 


Please refer to Section 13 of the IUCLID file for read-across documentation and rationale for the use of analogue chemicals for dihydromyrcenol.


 

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008


The available experimental test data are reliable but not sufficient for classification purposes according to Regulation (EC) No 1272/2008 (CLP), as amended for sixteenth time in Regulation (EU) No 2021/743.

Additional information