Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study in accordance with OECD TG 408 with restrictions. To address toxicological endpoints as part of the REACH registration of Benzyl Salicylate (Target Substance) it is proposed to read-across to Cyclohexyl Salicylate (Source Substance). The use of read-across works within the spirit of REACH and the stated aim of the legislation to reduce animal testing where possible. The Target Substance and Source Substance have been characterised using the categories and databases present in the OECD [Q]SAR Toolbox. From the profiling, it can be seen that the two substances share structural similarities and also ‘mechanistic action’ similarities which are both general and endpoint specific. Therefore, read across is justified.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
as of 12 May 1981
Deviations:
yes
Remarks:
The oral dose was only applied on five days a week
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
as of 1 March 1991, 91/325/EEC
Deviations:
yes
Remarks:
The oral dose was only applied on five days a week
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Cyclohexylsalicilate
- Physical state: colourless liquid
- Analytical purity: 100%
- Purity test date: not reported
- Lot/batch No.: 111 833 34/2
- Expiration date of the lot/batch: December 1995
- Stability under test conditions: stable in arachidis oil, DAB 10
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
other: Hsd/Win:Wu
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HarlanWinkelmann, Borchen, Germany
- Age at study initiation: about 5 weeks
- Weight at study initiation (at arrival): 51 to 65 g for males and 50 to 61 g for females
- Housing: two or three animals in Makrolon Type M 5 cages with standard softwood bedding
- Diet (e.g. ad libitum): pelleted Altromin Maintenance Diet 1324 ad libitum
- Water (e.g. ad libitum): community tap water ad libitum
- Acclimation period: 7 days under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 24
- Humidity (%): 39 to 65
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light (60 to 470 Lux)

IN-LIFE DATES: 109/110 days from the beginning of acclimatisation period (treatment groups) and 138 days from the beginning of acclimatisation period (recovery groups)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The solution of the test article and vehicle was prepared daily before administration, the solution was administered to the animals within approximately 2 to 3 hours after the preparation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): arachis oil, DAB 10; no justification for vehicle provided
- Concentration in vehicle: depending on dose the concentration was 8, 24 or 72 mg/mL (i.e. between 0.8 and 7.2%)
- Amount of vehicle (if gavage): the application volume was 5 mL/kg for all dose groups
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The nominal concentrations were analysed once by HPLC. The analysed concentrations were in good agreement with nominal concentrations: the dose of 40 mg/kg contained 0.415 g/50 mL ± 0.007 [nominal 0.4 g/50 mL], the dose of 120 mg/kg contained 1.265 g/50 mL ± 0.007 [nominal 1.2 g/50 mL] and the dose of 360 mg/kg contained 3.725 g/50 mL ± 0.049 [nominal 3.6 g/50 mL]
Duration of treatment / exposure:
102 to 103 days for treatment groups
131 days for recovery groups
Frequency of treatment:
Once daily on 5 days per week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
40 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
120 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
360 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10 females and 10 males in each dose group plus 5 females and 5 males used as recovery animals in the control and the high-dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were derived from the already available oral 28-day study
- Post-exposure recovery period in satellite groups: After 69 applications animals of the recovery groups had a treatment-free period of 29 days
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: acclimatisation period once daily, treatment period twice daily, recovery period once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: acclimatisation period once daily, treatment period twice daily, recovery period once daily

BODY WEIGHT: Yes
- Time schedule for examinations: arrival, once during acclimatisation, weekly during treatment and recovery period

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Frequency: weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Frequency: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at beginning and end of treatment
- Procedure: slit lamp microscopy, after instillation of a mydriatic agent into the eyes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after six weeks of treatment and at the end of treatment period
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after six weeks of treatment and at the end of treatment period
- Animals fasted: No data
- How many animals: all

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER: Bone marrow smears at scheduled necropsy
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The animals of the main groups and recovery groups were sacrificed by an overdose of ether and exsanguinated by cardial heart puncture at open thorax. All animals were submitted to full necropsy procedures: external surfaces, orifices, cranial cavity, external surface of the brain, thoracic, abdominal and pelvis cavities and viscera, carcass. The following organs were weighed: brain, testes, heart, liver, spleen, adrenals, kidneys, thymus.

Various organs were preserved for histopathology and fixated in 10% neutral formalin (except for eyes fixated in Davidson's fluid): adrenals aorta, bone marrow, brain, caecum, colon, duodenum, epididymis, eyes, heart, ileum, jejunum, kidneys, liver, lungs, lymph node (submandibularis), lymp node (mesenteric), lymph node (inguinalis), mammary gland, oesophagus, ovaries, pancreas, parathyroids, prostate, rectum, salivary glands, skeleta muscle, skin, spinal cord (lumbal region), spleen, stomach (fundus, pylorus), testes, thymus, thyroids, trachea, urinary bladder, uterus (horn, carvix), lumbal vertebrates, all gross lesions.

HISTOPATHOLOGY: Yes
Histopathology was performed on all fixated organs in the control group and the high-dose group.
Statistics:
T-test for determination of significant differences between the groups for body weight gains (L Sachs, Statistische Auswertungsmethoden, 3rd edition, Springer-Verlag, Berlin 1971); t-test for determination of significant differences between the groups for haematological and clinical chemistry results (CW Dunnett 1955, Journal of the American Statistical Association 372, 1096-1120 and 1964, Biometrics 1964, 482-490); Steel test for determination of significant differences between the groups for organ weights (RGD Steel 1959, Biometrics 15, 560-572)

Results and discussion

Results of examinations

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):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was increased in the high dose group.
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
Females, high dose group: significant, slight increase of the polymorphnuclear neutrophils in the differential white blood cell count, but within normal range of historical controls
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Males, high dose group: significant, slight decrease of bilirubin, but within normal range of historical controls
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Incidental, slight increase in liver weight
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Small areas of necrosis in the livers of 4 males in the high dose group, but similar findings also in male animals of other groups; therefore considered not treatment-related
Histopathological findings: neoplastic:
no effects observed

Effect levels

Dose descriptor:
NOEL
Effect level:
360 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: overall effects

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

To address toxicological endpoints as part of the REACH registration of Benzyl Salicylate (Target Substance)it is proposed to read-across to Cyclohexyl Salicylate (Source Substance).

The use of read-across works within the spirit of REACH and the stated aim of the legislation to reduce animal testing where possible.

The Target Substance and Source Substance have been characterised using the categories and databases present in the OECD [Q]SAR Toolbox. From the profiling , it can be seen that the two substances share structural similarities and also ‘mechanistic action’ similarities which are both general and endpoint specific.

See Section 13, document Read across justification_Cyclohexyl salicylate.

Applicant's summary and conclusion

Conclusions:
Repeated oral administration by gavage of the substance to female and male rats at doses ranging from 40 to 360 mg/kg bw/day on 5 days a week over a period of 90 days did not reveal adverse systemic effects. The NOAEL in this oral repeated dose toxicity study was 360 mg/kg bw/day.
Executive summary:

The oral repeated dose toxicity of the test substance, Cyclohexyl salicylate, over a period of 90 days was studied under GLP in accordance with OECD TG 408 with some restrictions. The test substance dissolved in arachis oil was administered to male and female rats by oral gavage on 5 days per week for a period of 90 days. Rats were about five weeks old at the initiation of the study and weighed between 50 and 65 g. The nominal test concentrations were 0 (vehicle control), 40, 120 and 360 mg/kg bw/day. The total volume of administered formulation was 5 mL/kg in all dose groups. In addition to the ten male and female rats per study group that were sacrificed at the end of the 90 -day treatment period, additional five male and female animals were used for investigation during a 29-day recovery period without oral treatment. The administration of test substance by oral gavage to male and female rats at doses of 40, 120 and 360 mg/kg body weight over a period of 90 days did not produce adverse systemic effects in this study.