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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 5, 2020 to September 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

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)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,3-dihydro-2,2,6-trimethylbenzaldehyde
EC Number:
204-133-7
EC Name:
2,3-dihydro-2,2,6-trimethylbenzaldehyde
Cas Number:
116-26-7
Molecular formula:
C10H14O
IUPAC Name:
2,6,6-trimethylcyclohexa-1,3-diene-1-carbaldehyde
Test material form:
liquid

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Crl:CD(SD) rats (SPF) was obtained from Charles River Laboratories Japan Hino Breeding Center.
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The vehicle and formulations were administered by gavage once a day using a syringe (Terumo) connected to a nelaton catheter (Terumo) at the amount of 5 mL/kg based on the latest body weights.
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed and olive oil was added to prepare the 2.50 w/v% formulation. A part of the 2.50 w/v% formulation was diluted with olive oil to prepare the 0.400 and 1.00 w/v% formulations.
The formulations and vehicle were subdivided into plastic containers, and stored at the cold place (target range: 1 to 10ºC). On each dosing day formulations and vehicle were taken out from the storage place and dosed to the animals. The formulations were used within 16 days after preparation.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable
- Mixing appropriate amounts with (Type of food): Not applicable
- Storage temperature of food: Not applicable

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item dissolved in olive oil at concentration of 20.0 w/v% (non-GLP). This vehicle is used for a general toxicity study and we have historical control data of this vehicle.
- Concentration in vehicle: Concentrations of the test item formulations were confirmed with HPLC in the first preparation (CERI study no. X02-0325). Actual concentrations of the 2.50, 1.00 and 0.400 w/v% formulations were 2.48, 0.980 and 0.400 w/v%, respectively. The formulations were within a permissible range of CERI Hita.
- Amount of vehicle (if gavage): Treatment volume was 5 mL/kg for control (negative, untreated group) and all treatment groups with applicable test item concentrations per group.
- Lot/batch no. (if required): lot no. 801029 and 810022, Japanese Pharmacopeia, TAISEI Pharmaceutical Industories
Duration of treatment / exposure:
Males and females were administrated for 14 days before mating. Thereafter, males were treated for 29 days including the mating period. Females were administrated until day 13 of nursing period throughout the mating and gestation periods. Non-mating females were treated for 29 days.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Recovery control group
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
Low - Group
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Intermediate - Group
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
High - Group
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
Recovery High - Group
No. of animals per sex per dose:
10 per sex per dose (10 male / 10 female) for mating groups; 5 animal per sex for control and high dose groups for recovery. For Each five animals of the first half are the main group, later five animals are the recovery group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a previously conducted 14-days sighting study (Report number from CERI, no. C21-0049).
- Rationale for animal assignment (if not random): Randomly assigned

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed for clinical signs twice a day, i.e. before and after dosing including observation of mortality during the dosing period. Females were observed delivery and nursing conditions. In the recovery period the animals were observed once a day.

BODY WEIGHT: Yes
Time schedule for examinations: Body weights were measured using an electric balance (SARTORIUS) on the following days:
- males on day 1, 3, 7, 14, 21, 28 of dosing and day 1, 7, 14 of recovery
- non-mating females on day 1, 3, 7, 14, 21, 28 of dosing and day 1, 7, 14 of recovery
- females on day 1, 3, 7, 14 of dosing, on gestation day 0, 7, 14, 20 and on postnatal day 0, 4, 8, 13, and day 21, 35, 42 and 49 of dosing for non-copulated female (animal no. 143)
- in addition, all animals were weighed on the necropsy days.

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

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from the abdominal aorta under isoflurane anesthesia from the following animals: five males and five dams for the same animals of the sensorimotor function examinations, non-mating five females, males and females of the recovery group, two pups per litter on day 4 after birth and two pups per litter at termination on day 13 after birth.
- Anaesthetic used for blood collection: Yes (isoflurane anesthesia)
- Animals fasted: Not specified
- How many animals: 39
- Parameters checked: Red blood cell count (RBC), Hemoglobin conc. (Hb), Hematocrit value (Ht), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin conc. (MCHC), Platelet count (Platelet), Reticulocyte count ratio (Reticulo), White blood cell count (WBC), Differentiation of leukocytes, Prothrombin time (PT) and Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected from the abdominal aorta under isoflurane anesthesia from the following animals: five males and five dams for the same animals of the sensorimotor function examinations, non-mating five females, males and females of the recovery group, two pups per litter on day 4 after birth and two pups per litter at termination on day 13 after birth.
- Animals fasted: Not specified
- How many animals: 39
- Parameters checked: Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (ALP), γ-Glutamyl transpeptidase (γ-GTP), Total cholesterol (T-Cho), Triglyceride (TG), Blood urea nitrogen (BUN), Creatinine, Total protein (T-Protein), Albumin, A/G ratio, Glucose, Total bilirubin (T-Bil), Total bile acids (TBA), Inorganic phosphorus (IP), Calcium (Ca), Sodium (Na), Potassium (K), Chloride (Cl) and Thyroid hormone (T4)

URINALYSIS: Yes
- Time schedule for collection of urine: Five males of the main group (same animals of sensorimotor function examinations), non-mating females, and males and females of the recovery group were housed in a metabolic cages in the afternoon of the each last observation day with free drinking and no feed until next morning for 15-17 h. Collected urines were examined for the following parameters. Urine sediments were examined in males and non-mating females in the control and high dose groups, and the recovery group.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: urine volume, color, turbidity, specific gravity, pH, protein, glucose, occult blood and urinary sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: last dosing week
- Dose groups that were examined: The animals were examined for the parameters of the following table in five males of each dose group, five non-mating females of the control and high dose groups, and selected five parental females of each dose group in each last dosing week.
- Battery of functions tested: reflex tests / grip strength / motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Liver, heart, kidneys, testes, epididymides, prostate, seminal vesicles, ovaries, uterus, brain, spleen, thymus, thyroid and adrenals were weighed using an electric balance (SARTORIUS) and their relative weights were calculated based on the body weight at the time of necropsy. Bilateral organs were weighed right and left together. Prostate was measured with a part of the urethra. Seminal vesicles were taken with coagulation gland after binding at the region of the origin. Thyroid adhered to the trachea including the parathyroid was fixed with 10% neutralized buffered formalin, and the right and left lobes were removed from the trachea and weighed on the next day of the necropsy. Uterus was measured containing enteral solution in non-mating females of the control and high dose groups and females of the recovery group.

HISTOPATHOLOGY: Yes
- Organs and tissues preserved in in 10% neutralized buffered formalin: Trachea, Lungs, Submandibular gland, Forestomach, Glandular stomach, Duodenum-ileum, Cecum- rectum, Pancreas, Liver, Heart, Kidneys, Urinary bladder, Testes, Epididymides, Prostate (ventral and
dorsolateral lobes), Coagulating gland, Seminal vesicle, Ovaries, Uterus (horn and cervix), Vagina, Cerebrum, Cerebellum, Pons, Spinal cord, Sciatic nerve, Bone marrow, Axillar lymph node, Mesenteric lymph node, Spleen, Thymus, Pituitary gland, Thyroid, Parathyroid, Adrenals, Organs and tissues, Eye ball, Skeletal muscle, Bone and Mammary gland.
Statistics:
Data regarding body weights of parental animals, food consumption, grip strength, locomotor activity count, parameters of hematology and blood chemistry, urine volume, urine specific gravity, organ weights, body weights on necropsy, mean estrous cycle length, pairing days until copulation, gestation length, number of implantation sites, number of pups born, number of live pups, body weights of pups, AGD and number of nipples/areolae were analyzed by the Bartlett’s test for homogeneity of variance. If significant difference (p<0.05) was not noted, the values of the control group and each test item group were analyzed by the Dunnet’s test. If significant difference (p<0.05) was noted in the Bartlett test, the values of the control group and each test item group were analyzed by the nonparametric Dunnet’s test.
Abnormal estrous cyclicity, copulation index, conception index and delivery dam index were analyzed by the Fisher’s exact test between the control group and each test item group.
Indexes of delivery, birth, viability and sex ratio were examined by the Bartlett’s test. If significant difference (p<0.05) was not noted, the values of the control group and each test item group were analyzed by the Dunnet’s test. If significant difference (p<0.05) was noted, the values of the control group and each test item group were analyzed by the nonparametric Dunnet’s test.
Data regarding body weights and food consumption during the recovery period, and parameters of hematology and blood chemistry, urine volume, urine specific gravity, organ weights and body weights at necropsy day for the recovery group were analyzed by the F-test for variance ratio. If there were no significant differences at a significance level of 5%, the Student t-test was performed. If there were significant differences at a significance level of 5%, the Aspin-Welch t-test was performed. The frequencies of defecation and urination during the recovery period were analyzed by the Mann-Whitney U-test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In males, salivation was observed just after dosing in all test item groups, and some animals salivated just before dosing in the 125 mg/kg group. One animal showed loosening and loss of right upper incisor in the 125 mg/kg group from day 27.
In females, salivation was observed in all test item groups, and some animals salivated just before dosing in the 50 and 125 mg/kg groups. Restlessness was observed just after dosing in almost animals of the 125 mg/kg group.

Salivation was observed in all test item groups. This change was considered related to the taste or smell of the test item since the animals transiently salivated just after or just before dosing, and was considered to be no toxicologically relevance. Restlessness was considered to be related to the salivation since the restlessness was observed as the same time as the salivation.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One animal (animal no. 123) in the 20 mg/kg group died during delivery (administration day 39).
One animal (animal no. 126) in the 20 mg/kg group showed much food on tray, decreased spontaneous locomotion, incomplete eyelids opening, staining around the anus, emaciation and reddish tear from postpartum day 4 (administration day 42), and this animal was sacrificed on postpartum day 8 (administration day 46) as a moribund animal.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption were increased in females of the 125 mg/kg group, this change was considered to be secondary effect of the salivation and no toxicologically significant.
Food efficiency:
no effects observed
Description (incidence and severity):
See body weight and weight changes sections.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in hematology or clinical biochemistry parameters.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in hematology or clinical biochemistry parameters.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related changes were noted in urinalysis except for renal tubular epithelial cells in males, which change was considered to be related to the histopathological lesions of the kidney.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In organ weights, relative weights of the kidneys were increased in males of the 125 mg/kg group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In macroscopic examinations, bilateral apparent spotty pattern of the surface of the kidneys was observed in males of the 50 and 125 mg/kg groups
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In histopathological examinations, accumulation of hyaline droplets in the proximal tubules of the kidney was observed in males of the 20, 50 and 125 mg/kg groups. This change was considered to be male rats specific and no toxicologically significant since relationship to α2u-globulin was confirmed. At the end of the recovery period, regeneration of the tubules of the kidney was observed in males of the 125 mg/kg group.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
urinalysis

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for males and females is defined as 125 mg/kg body weight per day in males and females.
Executive summary:

The study was performed according the requirements of OECD TG 422, under GLP conditions.

Male and female Crl:CD(SD) rats at 9 weeks of age were treated with the test item for 14 days and set on mating period for maximum of 14 days. Males and females were administrated until day 29 of dosing and 13 days after delivery, respectively. The dosage levels were set at 20, 50 and 125 mg/kg/day. Control animals were similarly dosed with olive oil.Recovery groups were set for the vehicle control and high dose groups.

The animals transiently salivated just after dosing or just before dosing in all test item groups. Restlessness was observed in females of the 125 mg/kg group.One female died in the 20 mg/kg group during delivery and other female showed moribund state after delivery, and these were considered to be no treatment-related. In urinalysis, renal tubular epithelial cells were observed in male of the 50 and 125 mg/kg groups.

There were no abnormal changes in detailed clinical observations, sensorimotor function examinations, body weights, food consumption, hematology or blood chemistry.

In organ weights, relative weights of the kidneys were increased in males of the 125 mg/kg group.

In macroscopic examinations, bilateral apparent spotty pattern of the surface of the kidneys was observed in males of the 50 and 125 mg/kg groups

In histopathological examinations, accumulation of hyaline droplets in the proximal tubules of the kidney was observed in males of the 20, 50 and 125 mg/kg groups.This change was considered to be male rats specific and no toxicologically significant since relationship to α2u-globulin was confirmed.

At the end of the recovery period, regeneration of the tubules of the kidney was observed in males of the 125 mg/kg group.

In conclusion, the No-Observed-Adverse-Effect Level (NOAEL) of Safranal P under the conditions tested was considered to be 125 mg/kg/day for repeated-dose toxicity