Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

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 (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2,3,4,4a,7,8,8a-octahydro-2,4a,5,8a-tetramethyl-1-naphthyl formate
EC Number:
265-742-1
EC Name:
1,2,3,4,4a,7,8,8a-octahydro-2,4a,5,8a-tetramethyl-1-naphthyl formate
Cas Number:
65405-72-3
Molecular formula:
C15H24O2
IUPAC Name:
2,4a,5,8a-tetramethyl-1,2,3,4,4a,7,8,8a-octahydronaphthalen-1-yl formate
Test material form:
liquid
Specific details on test material used for the study:
The test item will be Oxyoctaline Formate.
The following information refers to the original batch of test item received for the study:
Batch Number : SC00019275
Date of expiry : 02 January 2018
Appearance : Colourless to pale yellow liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Storage conditions : +4°C protected from light
Density : 1.0332 g/mL

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Assessment of the formulation for stability, homogeneity and concentration are regarded as unnecessary, the test item and control item being used in the form supplied.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
A total of 122 Hsd: Sprague Dawley SD rats (57 males and 65 virgin females), 7 to 8 weeks old and weighing 200 to 225 g for males and 175 to 200 g for females, will be ordered from Charles River Italia S.p.A., Calco (Lecco), Italy.
Sex:
male/female
Details on test animals or test system and environmental conditions:
The animals will be housed in a limited access rodent facility. Animal room controls will be set to maintain temperature and relative humidity at 22°C ± 2°C and 55% ± 15% respectively; actual conditions will be monitored, recorded and the records retained. There will be approximately 15 to 20 air changes per hour and the rooms will be lit by artificial light for 12 hours each day.
From arrival to allocation, animals will be housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5x38x20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material will be provided inside suitable bedding bags and changed at least twice a week.
From allocation to mating, males and females will be housed individually clear polysulfone cages measuring 42.5x26.6x18.5. Nesting material will be provided inside suitable bedding bags and changed at least twice a week.
During mating, animals will be housed one male to one female in clear polysulfone cages measuring 42.5x26.6x18.5 cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray will hold absorbent material which will be inspected and changed daily.
After mating, the males will be re-caged as they were before mating.
The females will be transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5x26.6x18.5 cm). Nesting material will be provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) will be provided as necessary. Nesting material will be changed at least 2 times a week.

Administration / exposure

Type of coverage:
semiocclusive
Details on exposure:
The test item or control item will be applied uniformly over the prepared skin area of animals.
Control animals will receive the control item at the highest dose level.
The dose will be administered to each animal on the basis of the most recently recorded body weight and the amount administered will be recorded for each animal.
The treated area will be covered using a piece of porous gauze covered with a self-adherent bandage.
An Elizabethan collar will be placed around the neck of each animal to prevent grooming of the treated site and oral ingestion of the test item or control item following dosing. The Elizabethan collars will remain in place for approximately 6 hours following dosing.
After a period of approximately 6 hours the Elizabethan collar and the bandage will be removed. The treated skin site will then be gently washed free of any remaining test item using 0.9% physiological saline.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Males
The amount of test item or control item will be adjusted once per week for each animal according to the last recorded body weight.
Animals will be dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, thereafter during pairing until Day 19 post coitum included. Dosing will be re-initiated starting from Day 4 post partum until Day 13 post partum or the day before sacrifice.
Females
The amount of test item or control item will be adjusted once per week for each animal according to the last recorded body weight.
During the gestation period, the amount will be calculated according to individual body weight on Days 0, 7, 14 and 19 post coitum and Days 4 and 7 post partum. Thereafter individual dose volumes will remain constant.
Frequency of treatment:
dosed once a day,
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
Dose / conc.:
800 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Each main group will comprise 10 male and 10 female rats (Groups 1 to 4).
Two groups (control and high dose levels) will include 5 animals per sex to be sacrificed after 2 weeks of recovery (Groups 5 and 6).
Control animals:
yes
Details on study design:
Individuals will be uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed and individually.
The cages will be identified by a label and recording the study number, animal numbers and details of treatment.
The arrangement of cages in batteries will be such that cages from each treatment group will be evenly distributed across the battery to minimise possible environmental effects.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions. Particular attention was paid on the treated site of animals (e.g. erythema and oedema)..
Sacrifice and pathology:
Parental animals in extremis, killed for humane reasons and those that have completed the scheduled test period and selected for blood collection, will be killed by exsanguination under isofluorane anaesthesia.
Parental males (Main groups)
The males will be killed after the mating of all females or after at least 28 days of treatment period.
Parental females (Main groups):
Statistics:
Standard deviations will be calculated as appropriate. For continuous variables the significance of the differences amongst group means will be assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings will be carried out by means of the non-parametric Kolmogorov-Smirnov test if n is more than 5.
The non-parametric Kruskal-Wallis analysis of variance will be used for the other parameters. Intergroup differences between the control and treated groups will be assessed by the non-parametric version of the Williams test. The criterion for statistical significance will be p<0.05.
Further tests will be used as considered appropriate. Details of all tests used and the data to which they are applied will be included in the Final Report.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The clinical signs including particular observation of the treatment site were performed daily after the removal of the bandage, approximately 6 hours after treatment start.
Only one male of the control groups showed moderate abrasion and hairloss on neck the day of sacrifice and one female tip of tail missing.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Dose level of 200mg/kg/day:
Males receiving 200 mg/kg/day showed: slight to moderate scabs, slight to marked erythema and slight to moderate oedema. These signs were evident on the treatment site, sometimes associated with fissuration and/or desquamation. The clinical signs appeared between Days 3 and 9. Abrasion on the treatment site was also noted in one male in a few occasions. Treated females showed slight to moderate scabs and erythema. Moderate desquamation was also noted. Apart from the local sign of irritation (erythema), noted in all females, the other clinical signs were observed in minor incidence compared to the treated males. The clinical signs appeared between Days 3 and 8. During the gestation period, the clinical signs noted in treated females were similar to those observed during the previous period. One female also showed slight oedema. From Day 20 post coitum to Day 3 post partum (when treatment stopped), no clinical signs were recorded and when treatment started again (from Day 4 to Day 13 post partum), scabs were noted in only one female.

Dose level of 400mg/kg/day:
Males receiving 400 mg/kg/day showed: slight to marked scabs, and slight to marked erythema. These signs were noted on the treatment site, sometimes associated to the presence of slight to moderate oedema. Fissuration and/or desquamation were associated to the primary signs of irritation. The clinical signs appeared between Days 3 and 9. Abrasion on the treatment site was also noted in two males. In one of these starting from Day 10 of treatment and in the other one as single occasion. Treated females showed slight to moderate scabs and slight to marked erythema. Slight desquamation, fissuration and slight oedema were also noted. The local sign of irritation (erythema) was observed in all females. The clinical signs appeared between Days 4 and 9. During the gestation period, the clinical signs noted in treated females were similar to those observed during the previous period, with a general trend of recovery. A general trend towards a recovery continued also from Day 20 post coitum to Day 3 post
partum (when treatment stopped) and thereafter when treatment started again (from Day 4 to Day 13 post partum).

Dose level of 800mg/kg/day:
Males receiving 800 mg/kg/day showed: slight to marked scabs and erythema. These signs were noted on the treatment site, sometimes associated to the presence of slight to moderate oedema. Fissuration and/or desquamation were associated to the primary signs of irritation. The clinical signs appeared between Days 3 and 9. Abrasion on the treatment site was also noted in 6 males starting from Day 10 of treatment Treated females showed slight to moderate scabs and slight to marked erythema. Slight to moderate desquamation, fissuration, slight to moderate oedema and slight abrasion were also noted. The local sign of irritation (erythema) was noted in all females. The clinical signs appeared between Days 4 and 10. During the gestation period, the clinical signs noted in treated females were similar to those observed during the previous period with a general trend of recovery. The secondary signs of irritation such as fissuration disappeared. A general trend towards a recovery continued also from Day 20 post coitum to Day 3 post partum (when treatment stopped) and thereafter when treatment started again (from Day 4 to Day 13 post partum). Fissuration and oedema disappeared.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Main groups
Means of bodyweight of males and femaleswere comparable to the control group throughout the study.
Statistically significant decreases in bodyweight gainwere noted in high dose males (800mg/kg/day) on Day 8 (-64%) and in all treated groups on Day 15 (- 21% to - 57%). Moreover, a statistically significant decrease was also noted in males of Group 3 (400 mg/kg/day) on Day 8 of the mating phase (- 34%). Occasionally, decrease in body weight gain was also observed in treated females of Group 3 on Day 7 post partum.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption measured in treated males and females, before pairing, during gestation and post partum, was comparable to the control group.
During treatment and recovery periods, no differences were noted in food consumption between control and treated group in both sexes receiving 800 mg/kg/day.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One male from each treated group (nos. X0440030, X0440048 and X0440078) showed neutrophilia. Compared with mean control data, changes were approximately 3.7 fold, with no dose-relation. Due to the minimal incidence and the absence of dose-relation, this change cannot be conclusively attributed to treatment. Some statistically significant changes were recorded in males dosed at 200 and/or 400 mg/kg/day (mean corpuscular haemoglobin concentration, platelets, reticulocytes). Changes were not dose-related, therefore they were considered incidental.

Coagulation - Dosing phase
Decrease of activated partial thromboplastin time was recorded in males receiving 400mg/kg/day. Due to the minimal severity and the direction, changes were considered not adverse, even though they could be treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Bile acids were increased in many treated animals. Compared with mean control data, the increments were 1.5 to 2.7 fold, with some dose-relation. In addition, male no. X0440048 (400 mg/kg/day) showed marked increases of alanine aminotransferase (4.2 fold), aspartate aminotransferase (8.5 fold), bilirubin (6.0 fold) and decrease of potassium (22%).
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Motor activity recorded at the end of treatment was comparable between control and treated groups. No toxicological significance was attributed to the change in the motor activity (increase) observed in males receiving 200 mg/kg/day, since it was dose unrelated. No relevant alterations in grip strength and sensory reaction to stimuli were observed in any treatment group at the examination performed at the end of treatment. Statistically significant increase in landing foot splay measurement (first trial), recorded as the individual and mean values of Group 3 females (400 mg/kg/day) was considered of no toxicological significance since it was not dose-related and observed in only one sex.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Terminal body weight was unaffected by treatment in both sexes, both in main and recovery groups. A slight but statistically significant decrease in terminal body weight was noted in Group 4 males (- 9%) compared to the control. Some statistically significant differences were noted in the organ weights (absolute and/or relative) in treated groups when compared to the control, such as:

– Decrease in absolute seminal vesicles (-18%) weight in males receiving 200mg/kg/day;
– Decrease in absolute (-28%) thymus weight in males receiving 800mg/kg/day;
– Increase in relative (+10%) brain weight in males receiving 800mg/kg/day;
– Increase in relative kidneys weight in all treated male groups ( +11%, +12%, +18%);
– Increase in relative (+22%) testes weight in males receiving 800mg/kg/day;
– Decrease in relative (-21%) thymus weight in males receiving 800mg/kg/day.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At post mortem examination, treatment-related changes were mainly noted in males in a dose related trend. The changes consisted of multiple scabs of the treated skin. In addition, red colour of the treated skin was observed in one treated male per group and two high dose treated females, when compared with controls. No other remarkable differences were noted at post mortem examination in treated animals, when compared with controls, either in animals that completed the treatment or recovery period. Gross lesions such as pelvic dilatation of kidneys in a high dose male (no. X0440062), and in low dose female (no. X0440029), enlarged ovaries in a high dose female (no. X0440075), enlarged inguinal lymph node noted in a single high dose male (no. X0440064) and reduced size of thymus in control and treated females were considered to be related to a physiological variation in pregnancy or to a spontaneous/incidental pathology.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
dermal irritation

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
400 mg/kg bw/day (nominal)
System:
other: Skin
Organ:
skin

Applicant's summary and conclusion

Conclusions:
Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered 400mg/kg/day for males and female.
Executive summary:

The NOAEL for general toxicity was considered 400mg/kg/day for males and female.