Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5th May 1994 to 31st May 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, equivalent to a valid guidelines and the study was conducted under GLP conditions. The study was performed with test material being used to support the substance on the basis of read-across.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to justification in seciton 13

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): CHS/Cyclohexylsalicilate
- Physical state: colurless liquid
- Storage condition of test material: room temperature
- Molecular formula (if other than submission substance): C13H16O3
- Molecular weight (if other than submission substance): 220.27
- Smiles notation (if other than submission substance): C(=O)(c1c(O)cccc1)OC1CCCCC1
- Structural formula attached as image file (if other than submission substance): see Fig. 1

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Wiga GmbH, D-97633 Sluzfeld, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: mean approximately 214 g
- Housing: Individually in Makrolon Type M3 cage
- Diet: pelleted Altromin Maintenance Diet 1324 ad libitum
- Water: tap water ad libitum
- Acclimation period: 5 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: arachadis oil
Details on exposure:
VEHICLE
- Concentration in vehicle: 0% (group 1), 0.8% (group 2), 2.4% (group 3) and 7.2% (group 4).
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test material in the vehicle was analysed once in the study. The concentrations of the test material in the vehicle were dtermined by HPLC the concentrations found are as follows:
0.4 g/50 mL (eq. 40 mg/kg) = 0.415 g/50 mL ± 0.007
1.2 g/50 mL (eq. 120 mg/kg) = 1.260 g/50 mL ± 0.014
3.6 g/50 mL (360 mg/kg) = 3.720 g/50 mL ± 0.014
Details on mating procedure:
Mated animals were provided by the supplier.
Duration of treatment / exposure:
Days 6 to 15 of the gestation period.
Frequency of treatment:
Daily
Duration of test:
Until day 20 post coitum
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
120 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
360 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (working days)

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: gross macroscopic examination of all maternal organs with emphasis on the uterus, uterine contents, position of the foetuses in the uterus and the number of corpora lutea.
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: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
The following statistical methods were used:
- if the variables could be assumed to follow a normal distribution, the Dunnett-test, based on pooled variance, was applied for the comparison between the treated groups and the control group.
- the Steel-test was applied when the data could not be assumed to follow a normal distribution.
- Fisher's exact test for 2x2 tables was applied if the variables could be dichotomised without the loss of information (Bonferroni-Holm-corrected).
Indices:
Body weight gains of the maternal animals from days 0-6, 6-16, 16-20 and 6-20 post coitum were calculated.

The individual placentae and the body weight gains of the foetuses were recorded.

For the reproduction data, group mean values were calculated on a per dam basis and a percentage group basis. Only dams with live foetuses on day 20 post coitum were included in the calculations.

Mean foetal weights were calculated from the individual foetal weights on a per litter basis/group.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Gross pathological findings:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No deaths occurred in the controls or any of the test groups.

No treatment-related symptoms were observed in all treatment groups.

Body weights of the pregnant females were essentially similar in all groups. Mean corrected body weight gain of groups 3 and 4 were statistically different when compared to the group 1.

No treatment-related differences were noted between the mean reproduction data of the groups 2 - 4 when compared to group 1. In groups 2-4, the sum of post-implantation loss was decreased (level 5%). In groups 2 and 4, the sum of the total embryonic deaths was decreased (level 5%) and in group 3 (level 1%). In the groups 2 and 4, the sum of the total foetuses was increased (level 5%) and in the group 3 (level 1%). These findings were considered to be incidental and within the normal range.

No macroscopic changes were noted in the dams of any of the groups.

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
360 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Visceral malformations:
effects observed, non-treatment-related
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The weights of the live foetuses exhibited no significant differences on a litter and individual basis i.e. mean weight between the control group and the treatment groups.

The weights of the placenta showed no significant differences between the control and treatment groups. The mean value of the uteri, including content, showed an increase in groups 2 -4 (level 5%).

The sex ratio of the foetuses was not affected by treatment with the test material.

No macroscopical findings were noted at external examination of the foetuses which were considered to be an effect of the treatment with the test material. In group 1, were noted a beginning hydrops, spina bifida, exencephalia, micrognathia and one foetus with paleness. In group 3 were noted a hydrops, missing tail and one foetus with missing mandibula, no orifice and two dead foetuses. In group 4 were noted two foetuses with one common placenta.

Visceral examination:

Group 1: 140 examined foetuses
9 hydronephrosis
12 ureter waved
9 ureter dilatation
2 bronchial tree dilated
1 runt
1 hydrocephalus internus

Group 2: 152 examined foetuses
21 hydronephrosis
12 ureter waved
9 ureter dilatation

Group 3: 152 examined foetuses
16 hydronephrosis
16 ureter waved
8 ureter dilatation
2 gut protrusion our of abdomen (artefact)

Group 4: 162 foetuses examined
12 hydronephrosis
2 ureter waved
5 ureter dilatation
1 hematoma periorbital
1 runt

The visceral examination of the preserved foetuses did not reveal and treatment-related abnormalities.

Skeletal examination of foetuses:
General observations:
The statistically significant differences were considered to be incidental. The findings concerning the ossification of the foetal skeleton showed no dose-relationship i.e. there was no indication for an abnormal ossification of the foetal skeleton, skull bones, sternebrae and coccygeal vertebrae in the treated groups compared to the controls.

Retardations:
No relevant findings in any of the groups.

Variations:
The statistical analysis of the findings sternebrae, incompletely ossified, ossified or abnormally ossified, does not indicate a dose-relationship i.e. no disturbance of the ossification of the sternebrae could be observed in any of the treated groups when compared to the controls. Concerning the number of ribs, the statistically significant difference was considered to be incidental.

Malformations:
Group 1 - one case (skull bones irregular placed, micrognathia, vertebrae (cervical/thoracal/lumbal/sacral) cleaved), coccygeal vertebrae non ossified.
Group 2 - no variations
Group 3 - two cases (micrognathia; skull bones partially ossified, vertebral column caudal non ossified)
Group 4 - no variations

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOEL
Effect level:
360 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOEL
Effect level:
360 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: embryotoxicity

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Summary of performance of mated females

 Group

 1

 2

 3

 4

 Dose (mg/kg)

 0

 40

 120

 360

 No. of mated females

24

 24

 24

 24

 No. of pregnant females

 23

 23

 23

 22

 No. of mortalities

 0

 0

 0

 0

 No. of females included in statistical analysis (females with live foetuses at termination)

 23

 23

 23

 22

 

Table 2: Mean differences in dam body weights

 Days post coitum

 

 Group (mg/kg)

 0 - 6 (% gain)

 6 - 16 (% gain)

 16 - 20 (% gain)

 6 - 20 (% gain)

 Corrected body weight gain (%)

 1 (0)

 49.0 (22.6)

 83.3 (31.4)

 56.1 916.1)

 139.3 (52.5)

 22.9

 2 (40)

 50.0 (23.2)

 86.8 (32.7)

 59.3 (16.9)

 146.2 (55.1)

 21.7

 3 (120)

 50.4 (23.4)

 83.2 (31.3)

 59.6 (17.1)

 142.8 (53.7)

 19.5

 4 (360)

 50.2 (24.2)

 80.0 (31.0)

 56.2 (16.6)

 136.2 (52.8)

 17.6

 

Table 3: Summary of dam body weights

 Day post coitum

 Group 1 (0 mg/kg)

 Group 2 (40 mg/kg)

 Group 3 (120 mg/kg)

 Group 4 (360 mg/kg)

 0

 216.6

 215.2

 215.6

 207.5

 6

 265.6

 265.2

 266.0

 257.7

 16

 348.9

 352.0

 349.2

 337.7

 20

 405.0

 411.4

 408.8

 393.9

 

Table 4: Summary of reproduction data (dams with live foetuses)

 

 Group 1 (0 mg/kg)

 Group 2 (40 mg/kg)

 Group 3 (120 mg/kg)

 Group 4 (360 mg/kg)

 Number of dams

 23

 23

 23

 22

 Corpora lutea

 382

 385

 378

 381

 Mean

 16.6

 16.7

 16.4

 17.3

 S.D.

 1.8

 2.3

 2.3

 1.9

 Implantation sites

 329

 346

 335

 333

 % of corpora lutea

 86.1

 89.9

 88.6

 87.4

 Mean

 14.3

 15.0

 14.6

 15.1

 S.D.

 2.1

 2.0

 2.3

 1.6

 Pre-implantation loss

 53

 39

 43

 48

 % of corpora lutea

 13.9

 10.1

 11.4

 12.6

 Post-implantation loss

 35

 17 #

 16 #

 19 #

 % of implantation sites

 10.6

 4.9

 4.8

 5.7

 Embryonic deaths: total

 35

 17 #

 14 ##

 19 #

 Embryonic resorptions

 34

 13

 11

 14

 % of implantation sites

 10.3

 3.8

 3.3

 4.2

 Mean

 1.5

 0.6

 0.5

 0.6

 S.D.

 1.6

 0.6

 0.8

 0.8

 Foetal resorptions

 1

 4

 3

 5

 % of implantation sites

 0.3

 1.2

 0.9

 1.5

 Mean

 0.0

 0.2

 0.1

 0.2

 S.D.

 0.2

 0.4

 0.3

 0.4

 Foetuses

 

 

 

 

 No. of dams

 23

 23

 23

 22

 Total foetuses

 294

 329 #

 321 ##

 314 #

 % of implantation sites

 89.4

 95.1

 95.8

 94.3

 Mean

 12.8

 14.3

 13.9

 14.3

 S.D.

 2.6

 2.1

 2.2

 1.9

 Live foetuses

 294

 329

 319

 314

 Dead foetuses

 0

 0

 2

 0

 Malformed foetuses

 1

 0

 2

 0

 % of foetuses

 0.3

 0.0

 0.6

 0.0

 Mean

 0.0

 0.0

 0.1

 0.0

 S.D.

 0.2

 0.0

 0.3

 0.0

 Sex of foetuses

 

 

 

 

 Total males

 132

 146

 154

 154

 % of foetuses

 44.9

 44.4

 48.0

 49.0

 Mean

 5.7

 6.3

 6.7

7.0

 S.D.

 2.1

 2.7

 2.2

 1.9

 Total females

 162

 183

 165

 160

 % of foetuses

 55.1

 55.6

 51.4

 51.0

Mean 

 7.0

 8.0

 7.2

 7.3

 S.D.

 1.5

 2.8

 1.8

 2.2

  Weights of live foetuses (litter basis)  

 

 

 

         

 Males and females

 

 

 

 

 N (litters)

 23

 23

 22

 22

 Mean

 4.1

 4.2

 4.6

 4.4

 S.D.

 0.7

 0.8

 0.9

 0.8

 Runts

 

 

 

 

 N (litters)

 3

 1

 2

 2

 Mean

 1.0

 1.0

 1.0

 1.0

 S.D.

 0.0

 0.0

 0.0

 0.0

 Males

 

 

 

 

 N (litters)

 23

 23

 23

 22

 Mean

 4.2

 4.3

 4.7

 4.5

 S.D.

 0.7

 0.8

 1.0

 0.8

 Runts

 

 

 

 

 N (litters)

 2

 0

 1

 0

 Mean

 1.0

 0.0

 1.0

 0.0

 S.D.

 0.0

 0.0

 0.0

 0.0

 Females

 

 

 

 

 N (litters)

 23

 23

 23

 22

 Mean

 4.1

 4.1

 4.5

 4.3

 S.D.

 0.7

 0.7

 0.9

 0.7

 Runts

 

 

 

 

 N (litters)

 1

 1

 1

 2

 Mean

 1.0

 1.0

 1.0

 1.0

 S.D.

 0.0

 0.0

 0.0

 0.0

 Weights of placenta (litter basis)

 

 

 

 

 Males and females

 

 

 

 

 N (litters)

 23

 23

 23

 22

 Mean

 0.6

 0.6

 0.6

 0.6

 S.D.

 0.1

 0.1

 0.1

 0.1

 Weights of uteri (dam basis)

 

 

 

 

 N (dams)

 23

 23

 23

 22

 Mean

 78.2

 89.2 *

 91.6 *

 90.7 *

 S.D.

 16.2

 11.7

 18.4

 14.1

# Fisher's Exact test (Bonferroni-Holm-corrected) significant at level 5% (#), 1% (##)

* Dunnett test based on pooled variance significant at level 1%

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test, the repeat oral administration of the test material to pregnant rats showed no symptoms of cumulative toxicity or any embryotoxic or teratogenic potential up to 360 mg/kg bw/day.
Executive summary:

The test material was tested at dose levels of 0 (group 1), 40 (group 2), 120 (group 3) and 360 (group 4) mg/kg bw. Each group consisted of at least 24 female rats (pregnant rats: 23 in groups 1 to 3 and 22 in group 4). The test material was administered orally by gavage daily from days 6 to 15 of gestation. A standard dose volume of 5 mL/kg bw was used. Control animals were dosed with the vehicle alone over the same period.

Clinical conditions and reactions to treatment were recorded at least once daily. Body weights were recorded for days 0, 6, 16 and 20 of gestation. All surviving females were sacrificed on day 20 of gestation and the foetuses were removed by caesarean section. At necropsy, the females were examined macroscopically and live foetuses were weighed, sexed and examined for visceral and skeletal abnormalities.

No mortality was observed at any of the dose levels tested. 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 necroscopy of the females. All females had viable foetuses. Pre-implantation loss, post-implantation loss, mean numbers of resorptions, embryonic deaths and total foetuses were not affected by treatment.

Mean foetal placental and uterus weights were not affected by treatment; foetal sex ratio was comparable in all group. No treatment-related foetal abnormalities were found at necroscopy; there were no treatment-related effects in the reproduction data. The examined foetuses showed no treatment-related malformations. The number of skeletal variations showed no treatment-related deviations. The number of skeletal ossifications in both the test groups and the control group were considered to be similar. The number of visceral variations in both the test groups and the control group were considered to be similar.

The results of the study show that repeat oral administration of the test material (days 6 - 15 post coitum) to pregnant rats caused no symptoms of cumulative toxicity up to 360 mg/kg bw/day. The test material reveals no embryotoxic or teratogenic potential at dose levels up to 360 mg/kg bw/day.