Registration Dossier

Administrative data

Description of key information

Read-across: 90-day oral repeated dose toxicity study : NOAEL = 30 mg/kg bw/day; LOAEL = 500 mg/kg bw/day (OECD 408, GLP, rel. 1)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Further information is included as attachment to the Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across approach is based on the hypothesis that the source and target substances have similar physico-chemical, (eco)toxicological and environmental fate properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and the source substances are both cyclohexenes branched with allyl ketone. The source substance differs in the allyl chain, by the position of the double bond and of the ketone functions, and an additional methyl group. The typical concentration and concentration range for the constituents and all identified impurities are shown in the attachment to the Iuclid section 13.

3. ANALOGUE APPROACH JUSTIFICATION
The source substance is expected to have similar toxicological profile than the target substance considering the respective physico-chemical and toxicological properties.
The study design (OECD 408, GLP) is adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the results of the screening for reproduction/developmental toxicity conducted in the rat with the source substance are likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VIII, 8.7.1.

4. DATA MATRIX
See Iuclid section13
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Remarks:
Read-across justification document
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
animals of either sex treated with 500 mg/kg bw/day showed a statistically significant increase in plasma creatinine, total protein and cholesterol as compared to control animals.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
animals treated with 500 mg/kg bw/day showed a statistically significant increase in liver and kidney weight (either sex); spleen weight (males).
Gross pathological findings:
no effects observed
Description (incidence and severity):
no macroscopic abnormalities were detected.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
(treatment related changes in liver, thyroid, bone marrow (at 500 mg/kg bw/day) and kidney (at 30 and 500 mg/kg bw/day))
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY:
- No mortality was observed. No clinically observable signs of toxicity were detected in test or control animals throughout the study period.
- Noisy respiration and increased salivation, together with associated findings of hunched posture and tiptoe gait were evident in 500 mg/kg bw/day treated animals throughout the treatment period. Incidents of increased salivation and noisy respiration were also evident in 5 and 30 mg/kg bw/day treated animals (respectively). These findings were not considered to represent systemic toxicity.
- At 30 and 500 mg/kg bw/day, females showed isolated episodes of tail elevation with the latter female also showing an episode of ataxia. The aetiology of these findings are unclear in the absence of any supporting behavioural assessments to suggest neurotoxicity and these changes were considered of no toxicological importance.
- Isolated instances of generalised fur loss, scabs formation or generalised red/brown stained fur were evident in a number of control and treated animals throughout the dosing period, however, these findings were considered to be entirely
incidental and unrelated to treatment.
- One control male showed episodes of noisy respiration whilst one control female developed hunched posture over a two day period. These findings were clearly unrelated to treatment.

BODY WEIGHT AND WEIGHT GAIN:
- No adverse effect on bodyweight development was detected. Bodyweight gain in test animals during the treatment period was similar to that of controls.
- Females treated with 500 mg/kg bw/day showed a statistically significant reduction in bodyweight gain during week 7 (p<0.05) together with an increase in bodyweight gain during weeks 9 and 13 (p<0.05 and p<0.001 respectively).
- Mean bodyweight at termination was at least 99 % of control and in the absence of any effect on overall bodyweight gain, differences in weekly bodyweight change were considered to reflect normal biological variation and to be of no toxicological significance.

FOOD CONSUMPTION AND FOOD EFFICIENCY:
- There was no adverse effect on food consumption during the entire study period. Food efficiency (the ratio of bodyweight gain to dietary intake) was similar to that of controls.

WATER CONSUMPTION:
- No intergroup differences were detected.

OPHTHALMOSCOPIC EXAMINATION:
- There were no treatment-related ocular effects. The incidental findings recorded were those normally encountered in laboratory maintained rats of this age and strain.

HAEMATOLOGY:
- No treatment-related changes in the haematological parameters were measured.

CLINICAL CHEMISTRY:
- Animals of either sex treated with 500 mg/kg bw/day showed a statistically significant increase in plasma creatinine, total protein and cholesterol (p< 0.01) compared to control animals. Males from this treatment group also showed a statistically significant increase in plasma albumin (p< 0.01). No such effects were detected in animals of either sex treated with 5 or 30 mg/kg bw/day.
- Animals of either sex treated with 500 mg/kg bw/day showed a statistically significant reduction in aspartate aminotransferase (p<0.05 males and p<0.001 females) whilst females also showed a reduction in alkaline phosphatase (p<0.05). Males treated with 500 mg/kg bw/day also showed a statistically significant reduction in plasma chloride concentration (p<0.05). Females from all treatment groups showed a statistically significant reduction in plasma bilirubin (p<0.05). The majority of individual values were within the normal range for rats of the strain and age used and in the absence of a dose related response the intergroup differences were considered of no toxicological importance.

NEUROBEHAVIOUR:
- No treatment-related changes in the behavioural, functional performance parameters and sensory reactivity were measured.

ORGAN WEIGHTS:
- Animals of either sex treated with 500 mg/kg bw/day showed a statistically significant increase in liver weight (p<0.01) both absolute and relative to terminal bodyweight. Males treated with 500 mg/kg bw/day also showed statistically significant increases in spleen and kidney weight (p<0.01) both absolute and relative to terminal bodyweight. Females treated with 500 mg/kg bw/day also showed a statistically significant increase in absolute and relative kidney weight (p<0.01). No such toxicologically significant effects were detected in animals of either sex treated with 5 or 30 mg/kg bw/day.
- Animals of either sex treated with 30 mg/kg bw/day showed a statistically significant reduction in absolute and relative heart weight (p<0.05). Females treated with 30 mg/kg bw/day showed a statistically significant increase in absolute and relative liver weight (p<0.05). These effects were considered to be of no toxicological importance.
- Males treated with 5 mg/kg bw/day showed a statistically significant increase in absolute and relative adrenal weight (p<0.05). In the absence of a dose-related response or any histological correlates this intergroup differences was considered not to be toxicologically significant.

GROSS PATHOLOGY:
- No macroscopic abnormalities were detected.

HISTOPATHOLOGY: NON-NEOPLASTIC
- LIVER: Hepatocyte enlargement, centrilobular or generalised, was observed in relation to treatment for 4/10 (minimal) males and 9/10 (minimal) females with 500 mg/kg bw/day (p< 0.05 for males; p< 0.001 for females). Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of associated degenerative changes may be interpreted as adaptive in nature.
- KIDNEYS: A greater incidence of higher severity grades of globular accumulations of eosinophilic material were observed in the tubular epithelium of 3/10 (minimal), 5/10 (slight), 1/10 (moderate) males treated with 500 mg/kg bw/day (p< 0.01) or in 5/10 (minimal), 4/10 (slight) males treated with 30 mg/kg bw/day (p< 0.05) but not at 5 mg/kg bw/day. The kidney changes identified histopathologically were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. Two males treated with 500 mg/kg bw/day and one male treated with 30 mg/kg bw/day exhibited higher grades of tubular basophilia.
- THYROID: A higher incidence of follicular cell hypertrophy was seen in relation to treatment for 7/10 (minimal) males treated with 500 mg/kg bw/day (p< 0.01).
- BONE MARROW: A higher incidence of lower grades of severity of adipose infiltration of the bone marrow, indicative of marrow hyperplasia, was observed in relation to treatment for 7/10 (minimal) and 3/10 (slight) males treated with 500 mg/kg bw/day (p< 0.01).

All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No treatment related effects were observed in females at 30 mg/kg bw/day.
Dose descriptor:
NOEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No treatment related effects were observed in males at 5 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Critical effects observed:
not specified

None

Conclusions:
Based on the read-across approach, the NOEL of the target substance is considered to be 30 mg/kg bw/day for females and 5 mg/kg bw/day for males and the NOAEL(combined) is considered to be 30 mg/kg bw/day based on histopathological changes in the kidney in male rats.
Executive summary:

In a repeated dose toxicity study performed in accordance with OECD test Guideline No. 408 and in compliance with GLP, test material in corn oil was administered by oral route (gavage) to three groups of Sprague-Dawley Crl:CD® (SD) IGS BR strain rats (10/sex/dose) at dose levels of 5, 30 and 500 mg/kg bw/day for 90 consecutive days. Control rats were given the vehicle alone. Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Following results were obtained:

 

- Mortality

No unscheduled deaths were observed.

 

- Clinical Observations

No clinically observable signs of toxicity were detected in test or control animals throughout the study period.

 

- Body weight

No adverse effect on bodyweight development was detected. Bodyweight gain in test animals during the treatment period was similar to that of controls.

 

- Food consumption

No adverse effect on dietary intake or food efficiency was detected.

 

- Water consumption

No intergroup differences were detected.

 

- Ophthalmoscopic examination

No treatment-related ocular effects were observed.

 

- Haematology

No treatment-related changes were detected.

 

- Clinical chemistry

Animals of either sex treated with 500 mg/kg bw/day showed a statistically significant increase in plasma creatinine, total protein and cholesterol (p< 0.01) compared to control animals. Males from this treatment group also showed a statistically significant increase in plasma albumin (p< 0.01). No such effects were detected in animals of either sex treated with 5 or 30 mg/kg bw/day.

 

- Neurobehavioral

No treatment-related changes in the behavioural, functional performance parameters and sensory reactivity were measured.

 

- Organ weights

Animals of either sex treated with 500 mg/kg bw/day showed a statistically significant increase in liver weight (p<0.01) both absolute and relative to terminal bodyweight. Males treated with 500 mg/kg bw/day also showed statistically significant increases in spleen weight (p<0.01) and in both sexes an increase in kidney weight (p<0.01) both absolute and relative to terminal bodyweight. No such toxicologically significant effects were detected in animals of either sex treated with 5 or 30 mg/kg bw/day.

 

- Gross pathology

No macroscopic abnormalities were detected.

 

- Histopathology

Liver: Microscopic examinations of liver sections revealed hepatocyte, centrilobular or generalised enlargement at 500 mg/kg bw/day. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of associated degenerative changes may be interpreted as adaptive in nature.

Kidney: Microscopic examination of kidney sections revealed globular accumulations of eosinophilic material in the proximal tubular epithelium of males treated with 30 and 500 mg/kg bw/day. The kidney changes identified histopathologically were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. Two males treated with 500 mg/kg bw/day and one male treated with 30 mg/kg bw/day also exhibited higher grades of tubular basophilia.

Thyroid: A higher incidence of follicular cell hypertrophy was observed in males treated with 500 mg/kg bw/day. Thyroxine is ultimately excreted via the bile, having first been conjugated in the liver. A hypothesis is that conjugating hepatic enzymes may have been induced as a response to the test material therefore increasing thyroxine excretion and stimulating compensatory thyroxine stimulating hormone and thyroxine production possibly resulting in the microscopic changes identified.

Bone marrow: A higher incidence of lower grades of severity of adipose infiltration of the bone marrow, indicative of marrow hyperplasia, was observed in relation to treatment in males treated with 500 mg/kg bw/day. This observation may suggest a subtle effect on haematopoiesis, lessened by the absence of supporting changes in the haematological parameters.

 

Based on the read-across approach, the NOEL of the target substance is considered to be 30 mg/kg bw/day for females and 5 mg/kg bw/day for males and based on histopathological changes in the kidney in males, the NOAEL(combined) is considered to be 30 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study is GLP-compliant and of high quality. The study was fully reliable (Klimisch score = 1).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No key study was available on the substance itself, therefore a read-across approach was used. The supporting substance, (3E)-3-methyl-4-(2,6,6-trimethyl-2 -cyclohexen-1-yl)-3-buten-2-one, is considered adequate for read-across purposes (see Iuclid section 13 for additional justification).

In the repeated dose toxicity study performed in accordance with OECD test Guideline No. 408 and in compliance with GLP (Safepharm, 2006, rel.1), test material in corn oil was administered by oral route (gavage) to three groups of Sprague-Dawley Crl:CD® (SD) IGS BR strain rats (10/sex/dose) at dose levels of 0 (olive oil), 5, 30 and 500 mg/kg bw/day for 90 consecutive days. No unscheduled deaths, no clinically observable signs of toxicity and no adverse effect on bodyweight development were observed. Bodyweight gain in test animals during the treatment period was similar to that of controls. No adverse effect on dietary intake or food efficiency was detected. No treatment-related ocular effects were observed. No treatment-related changes in the behavioural, functional performance parameters and sensory reactivity were measured.

Animals of either sex treated with 500 mg/kg bw/day showed a statistically significant increase in plasma creatinine, total protein and cholesterol (p< 0.01) compared to control animals. Males from this treatment group also showed a statistically significant increase in plasma albumin (p< 0.01). No such effects were detected in animals of either sex treated with 5 or 30 mg/kg bw/day.

Animals of either sex treated with 500 mg/kg bw/day showed a statistically significant increase in liver weight (p<0.01) both absolute and relative to terminal bodyweight. Males treated with 500 mg/kg bw/day also showed statistically significant increases in spleen weight (p<0.01) and in both sexes an increase in kidney weight (p<0.01) both absolute and relative to terminal bodyweight. No such toxicologically significant effects were detected in animals of either sex treated with 5 or 30 mg/kg bw/day. No macroscopic abnormalities were detected.

Microscopic examinations of liver sections revealed hepatocyte, centrilobular or generalised enlargement at 500 mg/kg bw/day. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of associated degenerative changes may be interpreted as adaptive in nature. Microscopic examination of kidney sections revealed globular accumulations of eosinophilic material in the proximal tubular epithelium of males treated with 30 and 500 mg/kg bw/day. The kidney changes identified histopathologically were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. Two males treated with 500 mg/kg bw/day and one male treated with 30 mg/kg bw/day also exhibited higher grades of tubular basophilia. A higher incidence of follicular cell hypertrophy was observed in males treated with 500 mg/kg bw/day. Thyroxine is ultimately excreted via the bile, having first been conjugated in the liver. A hypothesis is that conjugating hepatic enzymes may have been induced as a response to the test material therefore increasing thyroxine excretion and stimulating compensatory thyroxine stimulating hormone and thyroxine production possibly resulting in the microscopic changes identified. A higher incidence of lower grades of severity of adipose infiltration of the bone marrow, indicative of marrow hyperplasia, was observed in relation to treatment in males treated with 500 mg/kg bw/day. This observation may suggest a subtle effect on haematopoiesis, lessened by the absence of supporting changes in the haematological parameters.

The No Observed Adverse Effect Level (NOAEL) for repeated dose toxicity is therefore 30 mg/kg bw/day and the LOAEL is 500 mg/kg bw/day.

A supporting study was identified on the registered substance itself (Borriston, 1980, Rel.4). In this 14-day sub-acute toxicity study, test material was administered as a test diet for 14 days to the groups of CD® Sprague-Dawley outbred albino rats (5/sex/dose) at dose levels of 0 (plain diet), 250, 500, 1000 and 2000 mg/kg bw/day. Animals were observed for physical appearance, behaviour, mortality, body weight and food consumption. Animals were subjected to gross necropsy and liver and kidneys weighed. NOEL and LOAEL were considered to be 250 and 500 mg/kg bw/day, respectively, based on effects observed on mortality, clinical signs, body weight gain, food consumption.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008.

Self-classification:

Based on the available data on a supporting substance, no additional classification is proposed regarding the specific target organ toxicity after oral dose-repeated exposure.