Hexyl salicylate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 May 1994 - 2 February 1996.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

cyclohexylsalicylate

IUPAC Name:

cyclohexylsalicylate

Constituent 2

Reference substance name:

-

EC Number:

400-410-3

EC Name:

-

Cas Number:

25485-88-5

Molecular formula:

C13H16O3

IUPAC Name:

Cyclohexyl 2-hydroxybenzoate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Cyclohexylsalicylate
- Physical state: colourless liquid
- 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 or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Wiga GmbH, D-97633 Sulzfeld.
- Age at study initiation: 8 weeks
- Weight at study initiation: mean approximately 214g
- Fasting period before study: Not documented
- Housing: Housed individually in Makrolon Type M3 cage with standard softwood bedding.
- Diet (e.g. ad libitum): Pelleted Altromin Maintenance Diet 1324 ad libitum.
- Water (e.g. ad libitum): Community tap water from Dusseldorf ad libitum.
- Acclimation period: 5 days under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25°C
- Humidity (%): 44 - 75%
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light / 12 hours dark at lux units of 25 - 550.

IN-LIFE DATES: From: To: Not documented

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared daily before administration. The formulation was analysed and the concentrations of the test substance in arachis oil, DAB 10, based on the results of the determination of the HPLC method were:

0.8% = 0.4g/50ml (40 mg/kg) = 0.415 g/50ml ± 0.007
2.4% = 1.2g/50ml (120 mg/kg) = 1.260 g/50ml ± 0.014
7.2% = 3.6g/50ml (360 mg/kg) = 3.720 g/50ml ± 0.014

DIET PREPARATION
- Rate of preparation of diet (frequency): Daily
- Mixing appropriate amounts with (Type of food): Not relevant as gavage method of administration used
- Storage temperature of food: Not relevant as gavage method of administration used

VEHICLE
- Justification for use and choice of vehicle (if other than water): Arachidis oil, DAB 10.
- Concentration in vehicle: Not documented
- Amount of vehicle (if gavage): Not documented
- Lot/batch no. (if required): Not documented
- Purity: Not documented

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test concentrations were analysed using High Performance Liquid Chromatography.

Details on mating procedure:

No information on mating procedure provided as the rats obtained from the source were primiparous time-mated females.

Duration of treatment / exposure:

9 days treatment (from day 6 to day 15 post coitum)

Frequency of treatment:

Once daily

Duration of test:

20 days post coitum

No. of animals per sex per dose:

24 female rats per dose group
Group 1: 23 pregnant rats (0mg/kg)
Group 2: 23 pregnant rats (40 mg/kg)
Group 3: 23 pregnant rats (120 mg/kg)
Group 4: 22 pregnant rats (360 mg/kg)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The rationale for dose levels was based on the results of previous toxicological results.
- Rationale for animal assignment (if not random): Animals were randomly assigned.
- Other: No additional information.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily on working days

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule: Day 0, 6, 16 and 20 post coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: All maternal oragans, with emphasis on the uterus, uterine contents, position of foetuses in the uterus and the number of corpura lutea

OTHER:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: No data
- Other:

Fetal examinations:

- External examinations: Yes: all live foetuses
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

If the variables could be assumed to follow a normal distribution, the Dunnett-test based on pooled variance was applied for comparison between the treated and and control groups. The Steel-test was applied when the data could not be assumed to follow a normal distribution. Fisher's exact test for 2 x 2 tables was applied if the variables could be dichotomized without loss of information (Bonferroni-Holm-corrected).

Indices:

No information provided

Historical control data:

No information provided

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No compound related symptoms were observed in any treatment groups

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No deaths occurred in any of the test groups examined

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight profiles of the pregnant females were essentially similar in all groups. Mean corrected body weight gain of the treatment groups 3 and 4 were statistically different to group 1 (the vehicle control).

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic changes were noted in the dams of groups 1 - 4

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

In test groups 2 - 4, the sum of post-implantation loss was decreased by 5%, as was the sum of total embryonic deaths.The sum of embryonic deaths was also decreased by 1% in group 3. In groups 2 and 4, the sum of total foetuses was increased by 5% and in group 3 by 1%.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

not examined

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No deaths occurred in any of the test groups examined. No compound related symptoms were observed in any treatment groups. Body weight profiles of the pregnant females were essentially similar in all groups. Mean corrected body weight gain of the treatment groups 3 and 4 were statistically different to group 1 (the vehicle control). No compound related differences were noted between the mean reproduction data of the test groups in comparison to the control group. In test groups 2 - 4, the sum of post-implantation loss was decreased by 5%, as was the sum of total embryonic deaths.The sum of embryonic deaths was also decreased by 1% in group 3. In groups 2 and 4, the sum of total foetuses was increased by 5% and in group 3 by 1%. These findings were considered to be incidental and in the normal range. No macroscopic changes were noted in the dams of groups 1 - 4.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

360 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The weights of live foetuses exhibited no significant differences between the control group and the treatment groups. The weights of the placentae showed no significant differences between the control and the treatment groups. The mean value of the yteri including content showed an increase in the groups 2 - 4 (5%). The sex ratio of the foetuses was not affected by the treatment with the test article. On external examination, no macroscopic findings were noted which were considered to be an effect of the treatment with the test article. In the control group, hydrops, spina bifida, exencephalia, micrognathia and one foetus with paleness was observed. In the 120mg/kg treatment group, hydrops, missing tail and one foetus with missing mandibula, no orifice and two dead foetuses were observed. In the highest treatment group, it was noted that two foetuses had a common placenta.
On visceral examination, the following observations were noted:
Group 1 (control) : 140 foetuses examined - 9 foetuses displayed hydronephrosis
12 foetuses displayed waved ureters
9 foetuses displayed ureter dilatation
1 runt
1 foetus displayed hydrocephalous internus

Group 2 (40mg/kg) : 152 foetuses examined - 21 foetuses displayed hydronephrosis
12 foetuses displayed waved ureters
9 foetuses displayed ureter dilatation

Group 3 (120 mg/kg) : 152 foetuses examined - 16 foetuses displayed hydronephrosis
16 foetuses displayed waved ureters
8 foetuses displayed ureter dilatation
2 foetuses displayed gut protrusion out of the abdomen (considered an artefact)

Group 4 (360 mg/kg) : 162 foetuses examined - 12 foetuses displayed hydronephrosis
2 foetuses displayed waved ureters
5 foetuses displayed ureter dilatation
1 foetus displayed haematoma periorbital
1 runt

None of these visceral examinations revealed any treatment related abnormalities.
On skeletal examination, statistically significant differences were considered to be incidental. In all treatment groups, there were no relevant findings observed when retardation was examined. Any variations observed concerning ossification and the number of ribs where not considered to be statistically significant and were incidental. Skeletal malformations were observed in one case in group 1, with irregularly placed skull bones, micrognathia, cleaved vertebrae (cervical/thoracal/lumbal/sacral) and non-ossification of the coccygeal vertebrae. In treatment group 3, there was one case of micrognathia and one case of partial ossification of the skull bones and non ossification of the caudal vertebral column.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

360 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

No additional results.

Applicant's summary and conclusion

Conclusions:

The results of this study indicated that repeated oral administration of cyclohexylsalicylate to pregnant rats did not result in any signs of cumulative toxicity or any teratogenic or embryotoxic potential when tested to a dose level of 360 mg/kg body weight/day.

Executive summary:

In a study conducted by Pitterman (1996), the test substance, cyclohexylsalicylate, was examined for its ability to induce embryotoxic and teratogenic effects when administered to pregnant rats via oral gavage once daily from day 6 to day 15 post coitum. Four test groups were examined, with concentrations including 0 (control), 40, 120 and 360 mg/kg body weight/day. Each test group contained 24 rats.

Clinical condition and reaction to treatment were recorded at least once daily. Body weight was recorded on days 0, 6, 16 and 20 of gestation.

All surviving females were sacrificed on day 20 of gestation and the foetuses removed by caesarean section. Necropsy was performed and macroscopic examinations of the females conducted. Live foetuses were weighed, sexed and examined for visceral and skeletal abnormalities.

There were no mortalities observed at any dose level. No compound related symptoms were observed in any treatment group. Maternal weight gain was not affected by any treatment and no treatment related abnormalities were noted at necropsy. All females had viable foetuses. No treatment related foetal abnormalities were observed at necropsy and there were no effects on reproductive data.

The results of this study indicated that repeated oral administration of cyclohexylsalicylate to pregnant rats did not result in any signs of cumulative toxicity or any teratogenic or embryotoxic potential when tested to a dose level of 360 mg/kg body weight/day.