Dimethylcyclohex-3-ene-1-carbaldehyde

Endpoint summary

Description of key information

Vertoliff repeated dose information from a rat oral gavage OECD TG 407 study: NOAEL for systemic effects is 500 mg/kg bw.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

For repeated dose toxicity one subacute oral study is available performed with Vertoliff according to OECD guideline 407 in compliance with GLP.

System:

other: Increased liver weight

Organ:

liver

Additional information

Repeated dose toxicity – Endpoint summary

For Vertoliff a repeated dose toxicity study and a reproscreen study according to OECD TG 407 and OECD TG 421, respectively, are available, which are summarised below and an overall NOAEL of these two studies will be derived.

Vertoliff repeated dose toxicity in the OECD TG 407

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. Haematology 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.

Haematology 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 the substances seems to exceed the metabolic capacity).

Conclusion: the NOAEL for irritation is based on the clinical signs seen and the local irritation in the stomach at 750 mg/kg bw and is set to 500 mg/kg bw/day. The NOAEL for systemic effects is set to 500 mg/kg bw/day based on increased relative liver weights exceeding the 20% level. Other increased organ weights were seen and were without histopathological changes that indicated an adverse effect.

 

Vertoliff repeated dose toxicity seen in the Reproscreen toxicity study according to OECD TG 421

A reproduction/ developmental toxicity screening was performed according to OECD TG 421 and GLP.

Method: The substance was administered orally (gavage) to male and female Wistar Han IGS rats at dose levels of 50, 150 and 500 mg/kg bw/day (12 rats/sex/dose level). Concurrent controls (12 rats/sex) were performed with the vehicle (corn oil). Parental animals were exposed 2 weeks prior mating and during mating. Male rats and non-mated female rats were exposed until sacrifice (study day 30). Mated females were dosed from the start of the gestation period (the finding of a sperm positive vaginal smear was considered gestation day 0) up to delivery of the pups. During the lactation period, females were dosed up to sacrifice, after overnight fasting, on day 14 of lactation. The test substance was considered to be homogeneously distributed and to be stable in the gavage liquids under the experimental conditions. Although the concentration of the test substance of the mid-dose groups of the gavage liquids prepared was 14% lower than intended, in general, the concentration of the test substance was close to intended (90-110% of the intended concentration).

Results: For the male and female animals, there were no mortalities observed and no treatment related effects on macroscopic parameters and body weight were observed. Although food consumption was affected in the post-mating period of the male animals in mid- and high dose groups and in pregnant females in the high dose group, this was not considered as an adverse effect of the treatment. In male animals of the high-dose group, the absolute- (+13.6%) and relative (+16.8%) liver weights and the relative kidney weight (+8.0%) were statistically significantly increased as compared to the control animals. Microscopic evaluation revealed a treatment-related increase of accumulation of hyaline droplets in tubular epithelial cells in the outer cortex of the kidneys, accompanied by degenerative changes of these cells in 6/12 high dose males in most cases the hyaline droplets were also present in the lumen of the tubules. Immunohistochemically staining with a monoclonal antibody against alpha 2u revealed that there was insufficient evidence to identify the hyaline droplets as alpha 2 urinary microglobulins. In female animals of the mid- and high-dose groups, the absolute (+9.9% and +24.7%, respectively) and relative (+9.0% and +24.2%, respectively) weights of the liver were statistically significantly increased as compared to the control group. The increased liver weighs are somewhat high (>20%) but in absence of affected biochemistry parameters and histopathology these are considered non-adverse. No toxicological adverse effects on T4 and TSH hormones were observed in male and female parental animals.

In conclusion, systemic effects were observed in the high dose group (500 mg/kg bw/day). Though relative liver weight were slightly above 20%, these were not considered adverse in absence of biochemistry and histopathology changes. A NOAEL of 150 mg/kg bw/ day was therefore derived based on effects on kidney (male) and microscopic effects observed in the kidneys (male). For females the NOAEL was 500 mg/kg bw.

Repeated dose effects of Vertoliff in the OECD TG 407 versus the  OECD TG 421

In the table below the Vertoliff repeated dose effects from the OECD TG 407 and 421 are summarised to distinguish between the study report NOAELs and the overall NOAEL for repeated dose toxicity.

Local irritation effects: Irritation in the stomach is seen at the high dose of 750 mg/kg bw in OECD TG 407 but not in the OECD TG 421. The NOAEL for local effects is considered to be 500 mg/kg bw.

Systemic effects: Relative liver weight effects are seen at the high dose. In the OECD TG 407 study these weights increase up to ca 30% in both males and females. In the OECD TG 421 these are slightly lower ca 20% in males and females. In view of these increases at 30% in the OECD TG 407, these are considered adverse despite absence of related biochemistry values or histopathology in the liver and indicate overload of the metabolic pathway. The relative liver weights in the OECD TG 421 are around 20% and considered adaptive and not adverse.

Kidney: Some hyaline droplets related to alpha-2u globulin were seen in the OECD TG 407 in the control and high dose and therefore not considered adverse. In the OECD TG 421 these droplets were seen in the males in the high dose only but alpha-2u globulin could not be confirmed with the staining applied. The difference on the frequency and type of these droplets between the two studies makes the adversity questionable and were not taken into consideration for the overall NOAEL for repeated dose toxicity.  

Conclusion: The NOAEL for local irritation is set at 500 mg/kg bw/day based on clinical signs and irritant effects in the stomach at 750 mg/kg bw. For systemic effects most organ weights were not considered adverse due to absence of statistical significance, absence of dose relation and/or absence of histopathological findings. The NOAEL for systemic effects is based on the OECD TG 407 and set to 500 mg/kg bw/day based on increased relative liver weight up to ca 30% both in males and females indicating overloading the metabolic pathway.

Vertoliff	OECD TG 407	OECD TG 421
Strain of rat	Wistar	Wistar
	Key information	Key information
Doses	250/500/750	50/150/500
Effects
Clinical signs and functional tests	No treatment related effects	No treatment related effects
Heamatology and blood coagulation	No treatment related effects	No treatment related effects
Clinical biochemistry	No treatment related effects	No treatment related effects
Organ
Stomach histopathology	Irritation effects in stomach are seen in HD	No treatment related effects
Local NOAEL	500 mg/kg bw	500 mg/kg bw
Relative liver weights	Males and females, HD = +Ca 30%	Males +17 and +24% in females in HD
Liver histopathology	No treatment related effects	No treatment related effects
Relative kidney weights	No treatment related effects	Males only, HD = +8% (males only)
Kidney histopathology	All doses, in males: Hyaline droplets were confirmed to be alpha 2 u globulin present in control and high dose	In males: Hyaline droplets alpha-2uglobulin was not confirmed
Systemic NOAEL in report	750 mg/kg bw based on absence of histopathology effects in liver and other organs	150 mg/kg bw based on male kidney effects at 500 mg/kg bw
Overall assessment of systemic NOAEL mg/kg bw	500 based on increased relative liver weight > 20-30% (males and females)	500 relative liver weight increase is ca 20% and not considered adverse (males and females)

HD: High dose

Justification for classification or non-classification

The available repeated dose information on Vertoliff based on OECD TG 407 and 421 resulting in a systemic NOAEL of 500 mg/kg bw repeated dose classification of Vertoliff is not warranted in accordance with EU CLP (EC No. 1272/2008 and its amendments).