Registration Dossier

Administrative data

short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 March 1989 to 12 september 1989
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
according to guideline
EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
GLP compliance:
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
Cyclopentanone, 2-[(2R)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2S)-rel-
Molecular formula:
Cyclopentanone, 2-[(2R)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2S)-rel-
Constituent 2
Chemical structure
Reference substance name:
Cyclopentanone, 2-[(2S)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2R)-rel-
Molecular formula:
Cyclopentanone, 2-[(2S)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2R)-rel-
Constituent 3
Chemical structure
Reference substance name:
Cyclopentanone, 2-[(2S)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2S)-rel-
Molecular formula:
Cyclopentanone, 2-[(2S)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2S)-rel-
Constituent 4
Chemical structure
Reference substance name:
Cyclopentanone, 2-[(2R)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2R)-rel-
Molecular formula:
Cyclopentanone, 2-[(2R)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2R)-rel-
Test material form:
Specific details on test material used for the study:
Test substance Name (as stated in the report): NECTARYL - LRG 1371
Appearance: colourless, slightly viscous liquid
Batch number: AS 1241001
Storage: ambient temperature, away from light, air and heat

Test animals

Sprague-Dawley rats : Ico : OFA. SD. (IOPS Caw).
Details on species / strain selection:
- Supplier : : IFFA CREDO, Les Oncins, 69210 L'Arbresle, France.
- Justification: rodent species acceptable to the regulatory authorities with documented susceptibility to a wide range of toxic substances. Background data of the strain available at the testing facility
Details on test animals or test system and environmental conditions:
- Age at initiation of treatment : 9 weeks
- Body weight range at initiation of treatment: male (307 to 333 g) and female (195 to 245 g)
- Housing: one room for the study in an air-conditioned building (building K, barrier protected unit)
- Temperature: 19° to 26°C
- Humidity : 35 to 75 %
- Air changes: minimum 8 air changes per/hour
- Lighting cycle: 12 hours light {artificial) /12 hours dark
- Caging: animals housed singly in stainless steel mesh cages (260 x 350 x 200 mm)

Administration / exposure

Type of coverage:
unchanged (no vehicle)
The test article was administered as supplied
Details on exposure:
- Method of administration : the back and flanks of each animal were clipped from shoulders to rump to prepare a surface of approximately 40 cm2 ; the animals were clipped the day before the first application and twice a week thereafter.
The test article was applied pure on a surface of : 3 c m 2 in group 2 (about 1 % of the body surface), 10 to 12 crn 2 in group 3 (about 3 % of the body surface) and 35 to 40 c m 2 in groups 1 and 4 (about 10 % of the body surface) with the extremity of the syringe. The total shaved surface was covered with a double layer steril gauze held by a semi occlusive dressing (elastic bandage) during 5. 40 to 8 hours after this period, the application site was rinced with distilled water and the skin was dried with sterile absorbant cotton wool.
Duration of treatment / exposure:
4 weeks (28 administrations minimum). All animals were treated up to the day before necropsy (terminal ill at week 4).
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Group 1 Control (5 males and 5 females)
Dose / conc.:
50 mg/kg bw/day
Group 2 Low dose (5 males and 5 females)
Dose / conc.:
200 mg/kg bw/day
Group 3 Intermediaite dose (5 males and 5 females)
Dose / conc.:
1 000 mg/kg bw/day
Group 4 High dose (5 males and 5 females)
No. of animals per sex per dose:
5 males and 5 females per group / 1 dose concentration per group
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose selection: The high dose was the maximal dose recommanded by the OECD and EEC guidelines. The low dose was expected to be a non observable effect level. The intermediate dose was near to the geometric mean between high and low doses. The control animals received distilled water.


Observations and examinations performed and frequency:
- Morbidity/ mortality: All animals were examined twice daily at the beginning and at the end of the working day to detect any which were dead or moribund.
- Clinical signs: All animals were observed daily, before and at least once after administration during the treatment period, for signs of ill-health or adverse reaction to treatment. All full clinical examination was performed weekly.
- Local tolerance: Local tolerance was evaluated weekly according to the scale of evaluation in Addendum 3.
- Body weight: All animals were weighed weekly during the treatment period.
- Food consumption: Food consumption was recorded weekly for each cage during the treatment period.
- Clinical pathology (Techniques in Addendum 4): These investigations were performed for all animals at week 4 :
• Samples : Blood samples were withdrawn from the retro-orbital sinus following a light ether anaesthesia after an overnight fasting period of approximately 16 hours.
• Collection of samples: EDT A for haematology parameters, Trisodium citrate for prothrombin time, Tube without anticoagulant for biochemistry parameters .
• Parameters measured:

Haemoglobin level
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)
Packed cell volume
Red blood cell count
White blood cell count
Differential white blood cell count
Mean corpuscular volume
Platelet count
Prothrombin time

Inorganic phosphorus
Blood urea nitrogen
Total cholesterol
Total bilirubin
Total protein
Alkaline phosphatase
Aspartate aminotransferase (ASAT = SGOT)
Alanine aminotransferase (ALAT = SGPT)
Sacrifice and pathology:
Necropsy: All animals of each sex and each group were killed after 4 weeks of treatment following a randomisation. All animals were submitted to full necropsy procedures.
Animals were weighed, then killed by exsanguination following carbon dioxide inhalation.

Organ weights: The following organs were weighed: Adrenals, Testes, Liver, Kidneys, Brain.
Paired organs were weighed together. Organ weights expressed as an absolute value (g) and as an organ to body weigth ratio (g/100 g) and as an organ to brain weight ratio (g/g).

Organs/tissues preservation and histology: The following organs/ tissues were sampled for all animals :
Liver (1 section), Treated skin (1 section), Untreated skin (1 section) and Kidneys (2 sections).

Fixatives: All organs/tissues sampled were fixed and preserved in 10 % neutral formalin.

Staining: All organs/tissues were stained with haematoxylin and eosin.

Histopathological examinations: All org·ans/tissues of group 1 and 4 animals killed at week 4 were submitted to an histological examination.
For body weight, food consumption and clinical pathology parameters, in addition to the individual values obtained, the mean tables give for each criterion the mean (m) and standard deviation (s) calculated for each group.
All parameters were submitted to an analysis of variance (ANOVAR) which was performed to determine the intergroup variation and when it was statistically significant, the Dunnett's test was applied to determine which group was affected.
The statistical differences appear, under or beside the values, in tables as follows :
* p < o. 05
** p < 0.01
*** p < 0.001
For organ weights, the tables of mean values give for each different organ weighed :
the number or data (IN GRP)
the arithmetical mean (MEAN)
the standard deviation (STAND DEV)
the difference between the mean of the treated group and that of the control group (GROUP DIF).

For the treated groups, these results are followed by the results of the analysis of variance and the Dunnett's test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were observed.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Repeated dermal applications of the test article provoked erythema and desquamation at all dose levels. This local intolerance was dose-related and more marked in females than in males :
- in groups 2 and 3 , animals which were affected showed always a slight erythema or desquamation
- in group 4, 4 out of 5 males showed at least once a slight erythema and 3 out of 5 a slight desquamation whereas all the females showed a slight to moderate erythema and a slight to severe desquamation.
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight gain of treated groups was comparable to the control group values.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption of treated groups was comparable to the control values.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related variations were observed on haematological parameters examined.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In comparison with control group and after statistical analysis, the following variations were observed :
Calcium : slight decrease in group 3 males.
Total Bilirubin : slight increase in group 4 males.
These variations were not dose-related or the individual values of these parameters were within the normal limits of the strain. These variations could not be attributed to the treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed on organ weights .
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes observed at macroscopic examination.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At histological examination, minimal or moderate acanthosis with hyperkeratosis was observed in group 4 animals.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: No test item related changes observed, only slight local intolerance at the highest dose

Target system / organ toxicity

Key result
Critical effects observed:

Applicant's summary and conclusion

The NOAEL is found to be equal or greater than 1 000 mg/kg in this 28d dermal repeated toxicity study in rats.
Executive summary:

The objective of the study performed in 1989 according to the OECD Guideline No.410 was to evaluate the  toxicity of the test article  NECTARYL - LRG  1371  in the rat following dermal application for 4 weeks.

Four  groups  of  animals  were  placed  on  study  (Group 1 control: 0 mg/kg/day; Group 2 low dose 50 mg/kg/day; Group 3 intermediaite dose 200 mg/kg/day and Group 4 high dose 1000 mg/kg/day).

Clinical examinations were performed daily ; body weight,  food consumption and  local  tolerance  evaluation  were  performed,  weekly clinical pathology investigations were performed at week 4.              

At terminal kill, after macroscopic examination, selected organ were weighed, preserved and submitted to a histological  examination. No mortality was observed. No treatment-related abnormalities were observed in clinical condition or behaviour. Local  intolerance (erythema,  desquamation)  was observed  in  all treated groups with  a dose relationship,  more  marked  in  females than in males. Body weight gain and food consumption were not affected by treatment. No treatment-related  changes  were  observed  in  clinical  pathology.

No treatment-related changes were observed in organ weights and at macroscopic examination. At  histological  examination, a minimal to moderate acanthosis with hyperkeratosis  was observed  on treated  skin  in  group 4.  

The cutaneous lesions (erythema, desquamation acanthosis and hyperkeratosis) observed  in  the most of  treated animals and particularly at 1 000 mg/kg are frequently found in the rat after dermal applications of visquous test substance. They could not be considered  to be toxic effects  but  as slight local intolerance.The "no observable toxic effect level" is 1 000 mg/kg.