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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2021 - February 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Information was requested based on ECHA compliance check because of insufficient adequate read across from Triplal ( Cas no 27939-60-2 one of the isomers)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
October 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Dimethylcyclohex-3-ene-1-carbaldehyde
EC Number:
248-742-6
EC Name:
Dimethylcyclohex-3-ene-1-carbaldehyde
Cas Number:
27939-60-2
Molecular formula:
C9H14O
IUPAC Name:
(1R,6R)-3,6-dimethylcyclohex-3-ene-1-carbaldehyde; (1R,6R)-4,6-dimethylcyclohex-3-ene-1-carbaldehyde
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: e.g. Charles River, 97633 Sulzfeld, Germany
-- Age at study initiation: 7-8 weeks
- Weight at study initiation: males 284-335 gram; females 154-184 gram
- Fasting period before study: No
- Housing: Full barrier in an air-conditioned room
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days under laboratory conditions

DETAILS OF FOOD AND WATER QUALITY:
- Temperature: 22  3 °C
- Relative humidity: 55  10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on the test item’s characteristics and testing guideline.
- Concentration in vehicle: 10, 12.5, 15, 25, 35, 45, 60
and 75 mg test material / mL
- Amount of vehicle (if gavage): 4/ml/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Vertoliff was analysed in corn oil using GC-FID
Duration of treatment / exposure:
The animals were treated with the test item or vehicle on 7 days per week for a period of 28 days.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
40 animals (20 males and 20 females) were used for the study (5 male and 5 female animals per
group).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: At 750 mg/kg bw slight stomach irritation and histopathology effects were seen in an OECD TG 407 of a structural isomer (Triplal) but not in an OECD TG 421 with Vertoliff at 500 mg/kg bw. The irritation is anticipated due to the aldehyde functionality and/or the acidification of the aldehyde after oxidation. The effects at 750 mg/kg bw were considered tolerable but for animal welfare reasons the dose was not increased up to 1000 mg/kg bw.
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: overnight fasting yes
- Section schedule rationale: random:
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
All animals were observed for clinical signs during the entire treatment period of 28 days.
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge) and piloerection were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.
Ophthalmological examinations, using an ophthalmoscope were made on all animals before the first administration and in the last week of the treatment period.

BODY WEIGHT: Yes
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period.

FOOD CONSUMPTION: Food consumption was measured weekly during the treatment period.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmological examinations, using an ophthalmoscope were made on all animals before the first administration and in the last week of the treatment period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to or part of the sacrifice of the animals
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes: overnight
- How many animals:10/dose
- Parameters checked in table [No.3 or the report: section 11.14] were examined:

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to or part of the sacrifice of the animals
- Animals fasted: Yes: overnight
- How many animals: 10
- Parameters checked in table [No.5 of the report.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: prior to or part of the sacrifice of the animals
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes: overnight
- Parameters checked in table [No.6 of the report.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 8 in the report and below)

HISTOPATHOLOGY: Yes (see table 8 in the report and below)

Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using either a parametric oneway ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs: Slight to severe salivation was observed in 2/5 LD males and 1/5 LD females and in all mid dose and high dose animals. These animals also moved the bedding. These effects are considered to be effect of signs of local reactions indicative for the irritation of the substance and not of systemic toxicity.
Two males in the mid dose and all males and females in the high dose had slightly to severely reduced spontaneous activity which could be related to the test item (see page 42-50, as counted in the pdf).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Mortality: One animal in the low dose and one animal in the mid dose were euthanised in moribund condition because of misgavage (see page: 40 and 41 as counted in the pdf).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The test item had no effect on body weight development in this study. Overall the mean body weight increased normally during the observation period in control as well as in dose group animals. No statistically significant or dose-dependent differences between all male and female dose groups and the respective control group were found (see .pages 67 to 70 of the pdf report).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean food consumption of all male and female dose groups were comparable to control groups. No toxicologically relevant differences between the male and female dose groups and the respective control group were seen during the treatment period (See page 71 and 72 of the pdf report).
Food efficiency:
not specified
Description (incidence and severity):
Not relevant
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The test item had no toxicological relevant effect on haematological parameters and blood coagulation measured in this study.
Statistically significant changes were found in the females of the HD group in white blood cell count (WBC) (75.28% above control) and in the females of the LD and HD groups in red blood cell count (RBC) (9.06% and 7.88% above control, respectively). Haemoglobin (HGB) was slightly but statistically significantly elevated in females of the LD and MD groups when compared to the control group (7.14% and 7.84% above control). In females of all dose groups the haematocrit (HCT) was slightly but statistically significantly elevated (8.18%, 6.45% and 6.06% above control). The mean values for all these parameters were within the historical control data range for all dose groups. No dose-dependent difference to the control group occurred, the statistical significant changes in haematological parameters are considered to be not related to an effect of the treatment with the test item. Additionally, no differences with statistical significance were found for the male dose groups for any haematological parameter.
No statistical significance was noted for the coagulation parameters in any male and female dose group when compared to control group with the exception of aPTT in the male MD group (45.79% above control) and the male HD group (39.44% above control). The mean values for aPTT in males were within the historical control data range for all dose groups. Additionally, as the histopathological evaluation showed no test item-related effects and the statistical difference was not found in males, no toxicological relevance is considered. Furthermore, no test item-related effects were observed for the clinical pathological parameters (see page 73-76 as counted in the pdf report). Blood coagulation data are at page 77 and 78).
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The test item had no toxicological relevant effect on clinical biochemistry measured at the end of the treatment period.
At the end of the treatment period, statistical significance was seen for an increase in glucose (Gluc) and a decrease in cholesterol (CHOL) in males of the HD group when compared to the control group (39.50% above control and 22.95% below control, respectively). In females of the HD group, creatinine (Crea) was statistically significantly decreased (30.30% below control). The mean values for glucose, cholesterol and creatinine were within the historical control data range for all dose groups.
No statistically significant or toxicologically relevant differences were found for all other parameters in all male and female dose groups when compared to the control group (see page 79-82 as counted in the pdf report).
Endocrine findings:
no effects observed
Description (incidence and severity):
There were no effects seen in male and female reproductive organs).
Urinalysis findings:
no effects observed
Description (incidence and severity):
The test item showed no toxicologically relevant effects on all urinary parameters analysed on the day of necropsy (study day 29). No considerable differences with toxicological relevance were noted between the dose groups and compared to the control groups.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test item-related effects were observed in any parameter of the functional observation battery at the end of the treatment period when compared to observations before treatment.
In the last week of the treatment period, in males of the MD group, a slight but significant reduction in spontaneous activity was observed when comparing to control animals.
No ophthalmologic findings were observed in any of the animals of this study. The statistical significant difference was seen in the last week of the treatment period only in MD males. Therefore, no test item-related effect of the test item is considered (see pages 51 to 66 as counted in the pdf).
Immunological findings:
no effects observed
Description (incidence and severity):
There were no treatment related effect seen in any of the white blood cell parameters.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were no body or organ weight changes that could be related to the treatment with the test item at necropsy except for higher relative liver weight where a contribution of the test item cannot be excluded. However, in the absence of histopathological findings in the liver this is considered not adverse. No toxicologically relevant differences between the male and female dose groups and the respective control group were seen. Due to the low number of animals per sex and group a larger variability in absolute and relative organ weights is observed for some organs.
Relative organ to body weight ratio was statistically significantly higher for testes in the HD group when compared to the control group (~16% above control). As no histopathological changes have been observed in the testes and the value is within the historical control data, this increase is assumed to not be adverse. For testes the absolute weight is more important than the relative weight.
A statistically significantly higher absolute thyroid/parathyroid weight was recorded for males in the LD group when compared to control group males (~98% above control) as well as a statistically significantly higher organ to body weight ratio when compared to the control group males (~95% above control). Statistically significantly higher organ to brain weight ratio for thyroid/parathyroid was recorded for males in all dose groups when compared to the control group (~86%, ~58% and ~48% above control, respectively). The standard deviation for thyroid weight is rather high due to the very low number of animals per gender in each group in this study and can also be influenced by potential connective tissue that was not removed completely during necropsy. As only one gender is affected and this increase in thyroid weight is not accompanied by histopathological findings, this is not assumed to be adverse. In addition, all values are within the historical control data. There is also no dose related increase in weight.
Absolute liver weight was statistically significantly higher in males of the LD and HD groups and in females of the MD and HD groups when compared to the control group (males: ~12%, ~20%, females: 20% and ~28% above control, respectively). Relative organ to body weight and organ to brain weight ratios are statistically significantly higher for males and females in the HD group when compared to the control group (males: ~27% and 16%, females: ~33% and 29% above control, respectively). A dose-dependent trend towards higher relative liver weight (to body weight) was observed in both genders. Absolute and relative liver weights are at or slightly above the upper limit of the historical control data for the control and the dose groups. As histopathologically, in the HD group, besides haematopoietic infiltrations in single animals, there was no correlate, this is not assumed to be adverse.
A dose-dependent trend towards higher relative ovary weight (to body weight) was observed but was not statistically significant. The relevance of this trend is limited by the number of animals per group. This trend was not associated with any histopathological finding and therefore this is not assumed to be adverse.
A slight dose-dependent trend towards higher relative kidney weight (to body weight and to brain weight) was observed only in treated males. The relevance of this trend is limited by the number of animals per group. This trend was not associated with any histopathological finding and therefore this is not assumed to be adverse (see pages 83-92 as counted in the pdf report).
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic findings in animals euthanised for animal welfare reasons were in male no. 9 of the LD group a gelatinous beige mass on the ventral side of the thoracic cavity, which was filled with a clear fluid and a ruptured esophagus and in male no. 12 of the MD group a dark red and semi solid mass on the dorsal side of the stomach, a thickened urinary bladder and a fluid filled abdominal cavity.
In decedent animal no. 9 the findings recorded in the thoracic cavity correlate histopathologically with correlated with acute inflammation in the esophagus, mainly in the serosa. It caused a gelatinous mass in the thoracic cavity correlating to acute inflammation due to an amorphous mass deemed to be part of the test item.
The cause of death in male no. 12 was related to misgavage, which was considered to have caused a rupture of the stomach. Histologically, the gross lesions correlated with moderate acute inflammation and hemorrhage in the serosa. Other organs, e.g. spleen also showed serosal inflammation.
For the animals, sacrificed at the end of the treatment period, findings at necropsy were a brown semi-solid mass on the diaphragm (control male no. 1), small seminal vesicles of the left (MD male no. 11 and HD male no. 20), a red coloured thymus (LD female no. 28) and enlarged, red mandibular lymph nodes (LD female no. 26). These findings recorded at necropsy were considered incidental and were not related to the test item administration (see pages 93 t0 100 as counted in the pdf report).
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Stomach effects: Beside of the gross lesion-related serosal inflammation and hemorrhage in the decedent male no. 12, there was a minor increase of hyperkeratosis on the squamous epithelium in the forestomach in animals at 750 mg/kg bw/day. In addition, in one female at 750 mg/kg bw/day, there was a minimal ulceration in the forestomach associated with forestomach submucosal inflammation and hyperplasia of the squamous epithelium.
Kidneys: Hyaline droplets were seen in males in all dose groups. These were shown to be related to alpha-2uglobulin deposition by immune staining.
(The histopathology report is at page 176 to 310 as counted in the pdf; the summary tables start at page 193)

Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Remarks on result:
other: Highest dose tested; increased relative liver weights (ca 30%) which are considered adverse due to the magnitude of the change (> 20%)

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw (total dose)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
no
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw/day (actual dose received)
System:
other: liver
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
presumably yes

Applicant's summary and conclusion

Conclusions:
The local effect NOAEL for Vertoliff =500 mg/kg bw/day, based on clinical signs seen and slight stomach effects indicating that 750 mg/kg bw is the maximum tolerable dose in view of animal welfare.
The systemic NOAEL for Vertoliff = 500 mg/kg bw, the highest dose tested with treatment related increased relative liver, weight of 30% exceeding the (20% level which may be considered adaptive. Other organ weights thyroid, kidney and ovary weights were seen but without histopathological changes and therefore considered non-adverse.
Executive summary:

Introduction: The 28-day repeated dose toxicity of Vertoliff was tested in an oral gavage rat OECD TG 407 study. Repeated dose toxicity information of Vertoliff was available from an OECD TG 421 and a structural isomer Triplal (Cas no 27939-60-2 one isomer). These data indicated that the maximum high dose should be 750 mg/kg bw based on the irritancy seen at this dose in the stomach.  


Method: The test item was dissolved in corn oil and the control group was handled identically as the dose groups but received the vehicle instead of test item formulation. The groups comprised 5 male and 5 female Wistar rats each. The following doses were evaluated: 0, 250, 500 and 750 mg/kg bw. During the period of administration, the animals were observed precisely each day for signs of toxicity. Body weight and food consumption were measured weekly. Heamatology and clinical biochemistry parameter were measured. Animals that died were examined macroscopically and at the conclusion of the test, all surviving animals were sacrificed and observed macroscopically. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved. A full histopathological evaluation of the tissues was performed on high dose and control animals. Any gross lesion macroscopically identified was examined microscopically in all animals.


Results, mortality: There were two mortalities during the treatment phase. In both animals nos. 9 and 12 the cause of morbidity was most likely due to traumatic injury during the gavage procedure. Therefore, all lesions were considered incidental.


Clinical signs: There were clinical signs related to the irritancy of the substance e.g. slight to severe reduced spontaneous activity on some occasions, some in the mid dose and all males and females in the high dose. There were no test item-related clinical signs of systemic toxicity observed during the treatment period in any of the animals. In addition, detailed clinical examinations, functional observation battery (FOB) and ophthalmoscopy examination did not reveal any test item-related effects in any of the treatment groups.


Body weight and food consumption: In males and females, there was no test item-related effect on body weight or food consumption during the treatment period.


Heamatology and blood coagulation: No toxicologically relevant effects were observed in test item-treated animals.


Clinical biochemistry: No toxicologically relevant effects were observed in test item-treated animals.


Organs: In the stomach, there was a minor increase of hyperkeratosis on the squamous epithelium in the forestomach in animals at 750 mg/kg bw/day. In addition, in one female at 750 mg/kg bw/day, there was a minimal ulceration in the forestomach associated with forestomach submucosal inflammation and hyperplasia of the squamous epithelium. It is therefore considered that the test item caused a minor irritation at the high dose. Relative liver weights were increased at the high dose in males and females (ca 30%) but without histopathological changes. The magnitude of the change is considered adverse (> 20% as it seems to exceed the metabolic capacity).


Conclusion: the NOAEL for local irritation in the stomach is set 500 mg/kg bw/day. The NOAEL for systemic effects is set to 500 mg/kg bw/day based on increased relative liver weights. Other increased organ weights were seen and were withough histopathological changes that indicated an adverse effect.