Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1994-05-05 to 1994-05-31
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study according to OECD TG 414 on structural analogue, Cyclohexyl salicylate. 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:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
as of 12 May 1981
Deviations:
no
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%
- Impurities (identity and concentrations): not applicable
- 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:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 8 weeks, all animals were mated at the supplier and were delivered on gestation day 0
- Weight at study initiation: mean approximately 214 g
- Housing: individually in Makrolon Type M3 cages with standard softwood bedding
- Diet (e.g. ad libitum): pelleted Altromin Maintenance Diet 1324 ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 25
- Humidity (%): 44 to 75
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light (25 to 550 Lux)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The solutions of the test substance in arachidis oil were prepared daily before administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no justification provided
- Concentration in vehicle: depending on the dose level the concentration in the vehicle was 8, 24 or 72 mg/mL
- Amount of vehicle (if gavage): the formulations were administered at a constant volume of 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test substance in the solutions were analysed once. The concentrations were measured by HPLC at Henkel Analytical Center. The measured concentrations of the test substance in arachidis oil, DAB 10 agreed reasonably well with the nominal test 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 1.260 g/50 mL ± 0.014 [nominal 1.2 g/50 mL] and the dose of 360 mg/kg contained 3.720 g/50 mL ± 0.014 [nominal 3.6 g/50 mL]
Details on mating procedure:
Mating was performed at the supplier of the test system and details are not provided.
Duration of treatment / exposure:
From day 6 up to day 15 post coitum
Frequency of treatment:
Once daily
Duration of test:
On day 20 post coitum, the dams were sacrificed and foetuses were removed by Caesarean section
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:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: not reported

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: on day 0, 6, 16 and 20 post coitum

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: gross examination of all maternal organs with emphasis on uterus uterine content, position of foetuses in the uterus and number of corpora lutea, number of intrauterine implantations, late intrauterine deaths, early intrauterine; the uteri (including content) of all females were weighed at necropsy

OTHER: foetuses were sexed, weighed individually including placentae, examined for gross external abnormalities, half of the foetuses from each litter was fixed in Bouin's solution in order to examine viscera and brain by Wilson's slicing technique, remaining foetuses from each litter were placed in a solution of potassium hydroxide for clearing and were stained with alizarin red and skeletons were examined and preserved
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: No data
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
If the variables could be assumed to have a normal distribution, the Dunnett-test based on a pooled variance was applied for the comparison of the treated groups and the control group. The Steel test was applied when the variables were assumed to be not normally distributed. Fisher's Exact test for 2x2 tables was applied if the variables were dichotomised without loss of information (Bonferroni-Holm corrected).
Indices:
Pre-implantation loss, post-implantation loss, mean numbers of resorption, embryonic deaths and total foetuses

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No treatment related clinical signs were seen, body weight development was unaffected and there were no signs of adverse effects at necropsy. All females had viable foetuses. There was no effect on reproduction data.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
360 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Pre-implantation loss, post-implantation loss, mean numbers of resorptions, embryonic deaths and total foetuses were not affected by treatment. The foetal placental and uterus weights were not affected. Foetal sex ratio was comparable in all groups. No treatment related foetal abnormalities were found. The examined foetuses showed no treatment related malformations and figures on skeletal variations, skeletal ossifications and visceral variations were similar in all groups.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
360 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEL
Effect level:
360 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental 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 this 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:
Daily oral administration of the substance to pregnant Sprague-Dawley rats at doses of 40, 120 and 360 mg/kg bw/day from gestation day 6 to 15 did not cause maternal or foetal toxicity or teratogenic effects. The NOAEL in the study was 360 mg/kg bw/day.
Executive summary:

The potential embryotoxic and teratogenic effects of the substance cyclohexyl salicylate in the rat was studied under GLP in accordance with OECD TG 414. The substance was dissolved in arachidis oil and administered at a constant volume of 5 mL/kg body weight at doses of 0 (vehicle control), 40, 120 and 360 mg/kg bw/day to pregnant female rats from gestation day 6 to 15. All dams were sacrificed on gestation day 20 and the foetuses removed by Cesarean section. At necropsy the dams were examined macroscopically and foetuses were weighed, sexed and examined for visceral and skeletal abnormalities. The dams tolerated the applied dose levels up to 360 mg/kg bw/da without mortality. No compound related symptoms were observed in all treatment groups. Maternal body weight gain was not affected by treatment. No treatment related abnormalities were found at necropsy of the females. All dams had viable foetuses. Pre-implantation loss, post-implantation loss, mean numbers of resorptions, embryonic deaths and total number of foetuses were not affected by treatment. Mean foetal placental and uterus weights were not affected by treatment and the sex ratio was comparable in all groups. No treatment-related foetal abnormalities were found and there were no differences in the reproduction parameters between the groups. The examined foetuses showed no treatment related malformations. Skeletal variations and ossifications and visceral variations were similar in the control group and the treated groups. The NOAEL for maternal toxicity, embryotoxicity and teratogenicity was 360 mg/kg bw/day.