Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Screening for reproductive/developmental toxicity: Data waiving (study scientifically not necessary): According to REACH Annex VIII, column 2, the screening study does not need to be conducted because pre-natal developmental toxicity studies are available.

Extended one-generation reproductive toxicity: Data waiving (study scientifically not necessary): According to the column 1 of REACH Annex IX, the extended one generation reproductive toxicity study does not need to be conducted because the available repeated dose toxicity studies do not indicate adverse effects on reproductive organs or tissues nor reveal any other concern in relation with reproductive toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because a pre-natal developmental toxicity study is available
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
According to the column 2 of REACH Annex VIII, the study does not need to be conducted because pre-natal developmental toxicity studies are available.


Reason / purpose:
data waiving: supporting information
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
According to the column 1 of REACH Annex IX, the extended one generation reproductive toxicity study does not need to be conducted because the available repeated dose toxicity studies do not indicate adverse effects on reproductive organs or tissues nor reveal any other concern in relation with reproductive toxicity.
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Screening for reproductive/developmental toxicity: Data waiving (study scientifically not necessary): According to REACH Annex VIII, column 2, the screening study does not need to be conducted because pre-natal developmental toxicity studies are available.

Extended one-generation reproductive toxicity: Data waiving (study scientifically not necessary): According to the column 1 of REACH Annex IX, the extended one generation reproductive toxicity study does not need to be conducted because the available repeated dose toxicity studies do not indicate adverse effects on reproductive organs or tissues nor reveal any other concern in relation with reproductive toxicity.

Effects on developmental toxicity

Description of key information

Weight of evidence: Experimental results from pre-natal developmental studies performed with the components d-limonene, camphene, alpha terpinene and cineole are available:

Camphene: The NOEL for dams and fetuses was calculated to be 250 mg/kg bw/day. Also, as no teratogenic effects were observed at highest dose tested, a NOAEL >1000 mg//kg bw/day for developmental toxicity was established.

D-limonene in rats: The NOAEL for maternal toxicity was considered to be 591 mg/kg bw/day based on the deaths and decreased bodyweight gain. The NOAEL for fetal toxicity was considered to be 591 mg/kg bw/day based on the delayed skeletal formation and decreased bodyweight gain.

D-limonene in mice: The NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day based on the decreased bodyweight gain in dams and increased incidences of abnormal bone formation in fetuses.

D-limonene in rabbits: D-limonene was not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity was considered to be greater than 1000 mg/kg bw/day. The NOAEL for maternal toxicity was considered to be 250 mg/kg bw/day based on the decreased bodyweight gain.

Alpha terpinene in rats: The NOAEL of alpha terpinene for embryofoetal toxicity was determined to be 30 mg/kg body weight by the oral route based on reported signs of delayed ossification and a higher incidence of minor skeletal malformations observed at doses of 60 mg/kg bw or more. Nevertheless, the number of ossification centres affected in relation to the number examined was very small, even at the highest dose level tested indicating that the effect of treatment on the developing foetus was minimal. Also, it cannot be excluded that the observed maternal reproductive effects are secondary to general maternal toxicity. Thus, additional data would be needed to confirm the (absence of) observed effects and thus, these results should not contribute to the decision on classification of the substance (please see discussion on section "Justification for classification or not classification").

Cineole in rats: In a reproductive toxicity study performed by oral route (gavage) with 1,8-cineole using Wistar rats, the results obtained provide evidence that possibly the test substance presents maternal and fetal toxicity, but it is concluded that further studies need to be conducted. The NOAEL was not established since effects in maternal body weight were found at the lowest dose tested (250 mg/kg bw/day).

Conclusion: Experimental results from studies performed with the main components camphene and d-limonene have been selected in order to decide on the overall assessment of the test substance. Camphene was not teratogenic in rat fetuses and the NOAEL for fetal toxicity was considered to be greater than the highest dose tested. Also, d-limonene was not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity was considered to be greater than the highest dose tested. In two developmental toxicity studies with d-limonene in rats and mouse, slight ossification delays/malformations and organ weights changes were observed but not dose-related and/or observed at doses where maternal toxicity was identified. According to these results, the NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Prenatal developmental toxicity study: Groups of pregnant ICR mice (20/dose: 15 for teratogenicity study, 5 for postnatal development) were administered orally with d-limonene at dose levels of 0, 591 and 2363 mg/kg bw/day for 6 days from Day 7 to 12 of gestation and evaluated for developmental and postnatal development toxicity.
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
ICR
Details on test animals and environmental conditions:
No data
Route of administration:
oral: unspecified
Vehicle:
not specified
Details on exposure:
No data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Details on mating procedure:
no data
Duration of treatment / exposure:
6 days (gestation Day 7-12)
Frequency of treatment:
Once daily
Duration of test:
Gestatation Day 0 to postnatal week 7
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
591 mg/kg diet
Dose / conc.:
2 363 mg/kg bw/day
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Details on study design:
No data
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data
General behaviour observed, but no data regarding the frequency of observation

BODY WEIGHT: Yes
- Time schedule for examinations: daily

POST-MORTEM EXAMINATIONS: No data
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: No data
Examinations included: Number of implantations, number of resorptions and foetus bodyweight and placental weight
Fetal examinations:
- External examinations: Yes
- Visceral examinations: Yes
- Skeletal examinations: Yes
Statistics:
statistical significance difference of effects from controls were calculated at 5% and 1% levels.
Indices:
No data
Historical control data:
No data
Clinical signs:
no effects observed
Description (incidence and severity):
No anomalies were observed in the general behavior of dams during the period of gestation
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant decrease of bodyweight gain in pregnant mice was observed at 2363 mg/kg bw/day
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
Description (incidence and severity):
No anomalies were observed in the general behavior of dams during the period of gestation
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
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
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
591 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
A significant decrease of bodyweight gain was observed in male offsprings born to dams given drug orally at 2363 mg/kg bw/day, but there were not differences in weaning rate, sensory function, organ
weight and histological findings of the testis and ovary comparing with those of control.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
An incidence of lumber rib and fused rib in the fetuses increased significantly at 2363 mg/kg bw/day comparing with those of control.
In the observation of skeletal development in fetuses, retarded ossification of proximal phalanx of fore limb, metatarsal bone and proximal phalanx of hind limb were observed. However, these retarded
ossifications were restored to normal during postnatal development.
Visceral malformations:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
A significant decrease of bodyweight gain was observed in male offsprings born to dams given drug orally at 2363 mg/kg bw/day, but there were not differences in weaning rate, sensory function, organ
weight and histological findings of the testis and ovary comparing with those of control.
Key result
Dose descriptor:
NOAEL
Effect level:
591 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: rib
Description (incidence and severity):
Increase in incidence of lumber rib and fused rib compared to control.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
2 363 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
no
Relevant for humans:
not specified

Table 1: Effects of d-limonene on development of mouse fetuses.

 

Control

591

2363

No. of mothers

15

15

15

No. of implantations

162

179

154

(mean ± S.E.)

(10.80 ± 0.20)

(11.93 ± 0.13)

(10.27 ± 0.21)

No. of dead fetuses

9

1.1

6

(mean ± S.E.)

(0.60 ± 0.05)

(0.73 ± 0.10)

(0.40 ± 0.05)

No. of resorbed fetuses

18

20

20

(mean ± S.E.)

(1.20 ± 0.05)

(1.33 ± 0.10)

(1.33 ± 0.10)

No. of live fetuses

135

148

125

Sex ratio (Male/Female)

1.33

0.83

1.13

Fetuses Body weight (g)

Male (mean ± S.E.)

1.34 ± 0.02

1.24 ± 0.01

1.28 ± 0.02

Female (mean ± S.E.)

1.28 ± 0.02

1.22 ± 0.01

1.19 ± 0.02

Placental weight (mg)

Male (mean ± S.E.)

94 (2 ± 2.0)

86 ( 0 ± 1.7)

89 (3 ± 1.7)

Female (mean ± S.E.)

87 (8 ± 2.6)

81 (5 ± 1.9)

83 (5 ± 2.2)

External observation

No. of fetuses examined

135

148

128

No. of fetuses malformed

0

4

0

Cleft palate

0

4

0

Incidence (%)

0

2.7

0

Visceral observation

No. of fetuses examined

71

76

68

No. of fetuses malformed

4

4

3

Enlargement of foramen ovale

4

4

3

Incidence (%)

5.6

5.3

4.4

Table 2: Effects of d-limonene on skeletal development of mouse fetuses

 Dose (mg/kg bw)

Control 

591

2363

No. of fetuses examined

64

72

61

Variation

Lumbar rib (%)

17 (26.6)

12 (16.7)

28 (46.7) *

Cervical rib (%)

1 (1.6)

(5.6)

1 (1.7)

Fused rib (%)

0 (0)

0 (1)

5 (8.3) *

Crooked rib (%)

2 (3.1)

0 (1)

0 (0)

Asymmetry of sternebrae (%)

2 (1.7)

7 (9.7)

7 (11.7)

Fused sternebrae

0 (0)

1 (1.4)

1 (1.7)

No. of ossification

Sternebrae

5.99 ± 0.01

5.97 ± 0.02

5.94 ± 0.03

Fore limb

Metacarpal bone

8.00 ± 0

7.97 ± 0.03

8.00 ± 0

Proximal phalanx

7.50 ± 0.13

7.67 ± 0.16

6.87 ± 0.30 *

Middle phalanx

1.16 ± 0.23

2.14 ± 0.29 **

2.18 ± 0.29 **

Distal phalanx

9.08 ± 0.32

9.72 ± 0.20

9.13 ± 0.36

Hind limb

Metatarsal bone

10.00 ± 0

9.92 ± 0.05

9.80 ± 0.09 *

Proximal phalanx

8.12 ± 0.23

8.03 ± 0.23

7.23 ± 0.39 *

Middle phalanx

0.09 ± 0.09

0.33 ± 0.18

0.20 ± 0.11

Distal phalanx

9.47 ± 0.24

9.72 ± 0.20

9.30 ± 0.31

Caudal vertebrae

7.12 ± 0.95

6.50 ± 0.21

7.00 ± 0.25

* Significantly different from the control at 5% level.

** Significantly different from the control at 1% level.

Table 3: Effects of d-limonene on postnatal development of mouse offsprings

 

 

d-Limonene (mg/kg bw)

 

Control

591

2363

No. of mothers

5

5

5

No. of implantations

51

52

50

(mean ± S.E.)

(10.80 ± 0.33)

(10.41 ± 0.96)

(10.03 ± 0.63)

No. of offsprings

50

46

39

No. of dead offsprings at birth

0

0

0

Sensory function

Normal

Normal

Normal

No. of live offsprings

At birth

50

46

39

1st week

50

46

39

2nd week

50

46

39

3rd week

50

46

39

4th week

50

46

39

5th week

50

46

39

6th week

50

46

39

7th week

50

46

39

Weanling rate (%)

100

100

100

Table 4: Effects of d-limonene on gross differentiation of mouse offsprings

 

 

d-Limonene (mg/kg bw)

Gross differentiation

Control

591

2363

Opening of the ear-shell

3.5 ± 0.07

3.6 ± 0.08

4.1 ± 0.08

Coating with the hair

5.0 ± 0.00

4.11 ± 0.08

5.2 ± 0.06

Odontiasis

9.8 ± 0.07

9.3 ± 0.07

9. 1 ± 0.04

Opening of the eyelid

13.3 ± 0.08

12.9 ± 0.06

13.6 ± 0.09

Descending of the testis

23.0 ± 0.20

23.3 ± 0.11

25.0 ± 0.27

Opening of the vaginal orifice

29.5 ± 0.26

30.5 ± 0.16

30.6 ± 0.15

Table 5: Absolute organ weights of postnatal mouse offsprings born to mothers given d-limonene

Sex

Dose

(mg/kg bw)

No. of

offsprings

Final BW (g)

Thyroids (mg)

Thymus (g)

Lungs (g)

Heart (g)

Spleen (g)

Kidneys (g)

Liver (g)

Adrenals (g)

Testes (g) or Ovaries (mg)

Female

Control

27

34.2 ± 0.50

5.43 ± 0.18

75.72 ± 4.14

199.26 ± 4.70

158.22 ± 2.13

131.54 ± 9.96

0.63 ± 0.02

2.06 ± 0.09

8.39 ± 0.49

217.52 ± 1.65

591

27

34.9 ± 0.44

6.04 ± 0.68

65.26 ± 4.26

203.76 ± 2.95

166.66 ± 7.58

121.66 ± 4.74

0.63 ± 0.02

2.08 ± 0.07

8.29 ± 0.67

209.76 ± 9.23

2363

23

32.2 ± 0.77

5.41 ± 0.62

64.08 ± 3.64

189.60 ± 6.69

158.96 ± 3.12

125.52 ± 3.04

0.58 ± 0.02

2.14 ± 0.04

8.53 ± 0.86

200.20 ± 0.39

Male

Control

23

27.3 ± 0.50

4.80 ± 0.22

81.47 ± 4.38

172.80 ± 7.20

118.98 ± 3.91

121.76 ± 6.48

0.37 ± 0.01

1.37 ± 0.02

11.71 ± 0.40

13.38 ± 0.68

591

19

28.8 ± 0.45

4.29 ± 0.20

69.44 ± 5.52

174.26 ± 5.66

129.96 ± 3.62

112.68 ± 2.79

0.38 ± 0.01

1.37 ± 0.04

11.36 ± 0.43

16.98 ± 1.71

2363

16

28.1 ± 0.34

3.53 ± 0.41 *

63.95 ± 9.72

171.75 ± 5.70

135.15 ± 6.89

116.15 ± 5.78

0.38 ± 0.01

1.46 ± 0.03 *

11.61 ± 0.30 *

17.93 ± 1.30 *

* Significantly different from the control at 5% level.

Table 6: Relative organ weights per 100 g body weights of postnatal mouse offsprings born to mothers given d-limonene

Sex

Dose

(mg/kg bw)

No. of

offsprings

Final BW (g)

Thyroids(mg/100 g)

Thymus(mg/100 g)

Lungs(mg/100 g)

Heart(mg/100 g)

Spleen(mg/100 g)

Kidneys(g/100 g)

Liver(g/100 g)

Adrenals(mg/100 g)

Testes or Ovaries (mg/100 g)

Female

Control

27

34.2 ± 0.50

15.80 ± 0.44

220.36 ± 10.67

580.50 ± 13.59

461.18 ± 8.65

386.55 ± 39.35

1.83 ± 0.07

6.01 ± 0.26

24.48 ± 1.55

634.84 ± 19.06

591

27

34.9 ± 0.44

17.20 ± 1.93

185.29 ± 8.48 *

581.21 ± 9.42

474.60 ± 717.10

346.85 ± 12.29

1.80 ± 0.05

5.93 ± 0.14

23.55 ± 1.48

596.69 ± 14.95

2363

23

32.2 ± 0.77

16.55 ± 2.64

191.59 ± 6.25

568.43 ± 13.67

477.39 ± 11.90

376.91 ± 10.23

1.73 ± 0.03

6.44 ± 0.29

25.74 ± 2.82

602.30 ± 34.26

Male

Control

23

27.3 ± 0.50

17.13 ± 18.47

291.98 ± 34.66

619.50 ± 8.54

425.00 ± 32.37

437.67

1.31 ± 0.04

4.89 ± 0.08

41.89 ± 1.41

47.85 ± 2.42

591

19

28.8 ± 0.45

15.06 ± 0.61 *

242.35 ± 13.57

611.98 ± 17.12

456.02 ± 4.93 *

397.44 ± 20.40

1.33 ± 0.02

4.81 ± 0.11

39.96 ± 1.73

59.59 ± 5.78

2363

16

28.1 ± 0.34

12.60 ± 1.44 *

229.38 ± 35.46

612.74 ± 20.32

482.14 ± 24.21 *

414.30 ± 17.34

1.37 ± 0.04

5.23 ± 0.09 *

41.45 ± 1.27

64.33 ± 5.38 *

* Significantly different from the control at 5% level.

Table 7: Summaried data on postnatal development of mouse offsprings

 

 

d-limonene (mg/kg bw)

 

Control

591

2363

External observation

 

No. of offsprings examined

50

46

39

No. of offsprings malformed

0

0

0

Visceral observation

 

No. of offsprings examined

50

46

39

No. of offsprings malformed

0

0

0

Skeletal observation

 

No. of offsprings examined

50

46

39

No. of offsprings malformed

0

0

0

Skeletal variation

 

Lumbar rib (%)

17 (34.0)

23 (50.0)

20 (51.3)

Fusion of 13th and lumbar rib (%)

0

0

1 (2.6)

Fusion of lumbar vertebra (%)

1 (2.0)

1 (2.2)

1 (2.6)

Crooked tail (%)

0

0

1 (2.6)

Conclusions:
Under the test conditions, the NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day based on the decreased bodyweight gain in dams and increased incidences of abnormal bone formation in fetuses.
Executive summary:

In a prenatal developmental toxicity study, d-limonene was administered orally to groups of pregnant ICR mice (20/dose: 15 for teratogenicity study, 5 for postnatal development) at dose levels of 0, 591 and 2363 mg/kg bw/day for 6 days from Day 7 to 12 of gestation. Bodyweights of pregnant mice were recorded during organogenesis. Caesarean sections were performed and the number of dead, live or resorbed fetuses, sex ratio and number of implantation sites were recorded. Fetuses were weighed and examined for external, visceral and skeletal malformations. Number of live offsprings, sensory functions, gross differentiation and organ weights of offsprings were recorded until postnatal week 7.

 

A significant decrease of bodyweight gain in pregnant mice was observed at 2363 mg/kg bw/day. However, no anomalies were observed in the general behavior of dams during the period of gestation. An incidence of lumber rib and fused rib in the fetuses increased significantly at 2363 mg/kg bw/day comparing with those of control. In the observation of skeletal development in fetuses, retarded ossification of proximal phalanx of fore limb, metatarsal bone and proximal phalanx of hind limb were observed. However, these retarded ossifications were restored to normal during postnatal development. A significant decrease of bodyweight gain was observed in male offsprings born to dams given drug orally at 2363 mg/kg bw/day, but there were not differences in weaning rate, sensory function, organ weight and histological findings of the testis and ovary comparing with those of control.

 

Under the test conditions, the NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day based on the decreased bodyweight gain in dams and increased incidences of abnormal skeletal formation in fetuses at 2363 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Prenatal developmental toxicity study: Groups of pregnant Japanese white rabbits were administered orally with d-limonene at dose levels of 250, 500 and 1000 mg/kg bw/day for 13 days from Day 6 to 18 of gestation and evaluated for teratogenicity.
GLP compliance:
not specified
Limit test:
no
Species:
rabbit
Strain:
other: Japanese white
Details on test animals and environmental conditions:
no data
Route of administration:
oral: unspecified
Vehicle:
not specified
Details on exposure:
no data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Details on mating procedure:
No data
Duration of treatment / exposure:
13 days (gestation Day 6-18)
Frequency of treatment:
Once daily
Duration of test:
Gestatation Day 0 to postnatal Day 49
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
10 (in 0-500 mg/kg bw/day groups) or 18 (in 1000 mg/kg bw/day group) pregnant females
Control animals:
yes
Details on study design:
no data
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

General behaviour observed, but no data regarding the frequency of observation

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
mean daily food consumption by treatment group is reported

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


POST-MORTEM EXAMINATIONS: No data

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: No data
Examinations included: Number of implantations, number of resorptions and foetus bodyweight and placental weight
Fetal examinations:
- External examinations: Yes: about 90% per litter
- Visceral examinations: Yes: about 90% per litter
- Skeletal examinations: Yes: about 90% per litter
Statistics:
statistical significance difference of effects from controls were calculated at 5% and 1% levels.
Indices:
no data
Historical control data:
no data
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No anomalies were observed in the general behaviour of dams given 250 and 500 mg/kg of d-limonene during the gestation.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Treatment with the highest dose level (1000 mg/kg) of d-limonene resulted in death of dams with less than 40% mortality.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The significant decrease of body- weight gain was temporarily observed in dams given 500 and 1000 mg/kg of d-limonene
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The significant decrease of food consumption was temporarily observed in dams given 500 and 1000 mg/kg of d-limonene
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:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
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
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral and skeletal examinations revealed some anomalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae. These did not appear to be dose-dependent and restored to normal during the postnatal development.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral and skeletal examinations revealed some anomalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae. These did not appear to be dose-dependent and restored to normal during the postnatal development.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Other non specific anomalies involved the lumber ribs in fetuses and offsprings, formation of the accessory ossification center of the 5th sternebrae in offsprings and the atrial septal defect detected in only 2 fetuses of a litter from dams treated with 250 mg/kg bw/day of d-limonene.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1: Effect of d-limonene on prenatal development of rabbit fetuses

Dose (mg/kg bw)

Control

250

500

1000

No. of pregnant animals

10

10

10

18

No. of dead clams

0

0

0

6

(%)

 

 

 

33

No. of examined clams

10

10

10

10

No. of implantations

96

94

85

91

(mean ± S.E.)

9.50 ± 0.25

9.40 ± 0.21

8.50 ± 0.33

9.10 ± 0.25

No. of resorbed fetuses

5

4

4

8

No. of dead fetuses

3

5

0

3

No. of live fetuses

88

85

81

80

Sex ratio (Male/Female)

0.73 (37/51)

1.13 (45/50)

0.62 (31/50)

1.11 (38/42)

Fetus body weight (g)

 

 

 

 

Male (mean ± S.E.)

44.39 ± 1.33

48.09 ± 1.07 *

44.76 ± 1.51

43.22 ± 0.96

Female (mean ± S.E.)

45.64 ± 1.00

47.45 ± 1.08

46.14 ± 1.21

45.13 ± 1.10

Placental weight (g)

 

 

 

 

Male (mean ± S.E.)

5.76 ± 0.17

5.84 ± 0.17

5.95 ± 0.29

5.77 ± 0.19

Female (mean ± S.E.)

5.87 ± 0.19

5.70 ± 0.15

6.16 ± 0.18

5.87 ± 0.23

* Significantly different from the control at 5% level

Table 2: Prenatal examinations of rabbit fetuses

Dose (mg/kg bw)

Control

250

500

1000

External examination

 

 

 

 

No. of examined fetuses

91

90

81

83

No. of malforrned fetuses

0

0

0

0

Visceral examination

 

 

 

 

No. of examined fetuses

88

85

81

80

No. of malformed fetuses

 

 

 

 

Atrial septal defect (%)

0

2 (2.4)

0

0

No. of minor abnormality

 

 

 

 

Incomplete lobulation of lungs (%)

11 (12.5)

16 (18.8)

19 (23.5)

19 (23.8)

Enlargement of foramen ovale (%)

2 (2.3)

2 (2.4)

5 (6.2)

4 (5.0)

Skeletal examination

 

 

 

 

No. of examined fetuses

86

87

81

80

No. of malformed fetuses

0

0

0

0

No. of variation

 

 

 

 

Left lumbar rib (%)

18 (20.9)

26 (29.9)

14 (17.3)

25 (31.3)

Right lumbar rib (%)

16 (18.6)

22 (25.3)

14 (17.3)

22( 27.5)

Ossification pattern

 

 

 

 

Retarded ossification of 5th sternebrae (%)

11 (12.8)

14 (16.1)

8 (9.9)

18 (22.5)

Retarded ossification of middle phalanx of fore limbs (%)

2 (2.3)

3 (3.4)

0

6 (7.4)

Table 3: Absolute organ weights of rabbit offsprings

 

Male

Female

 

Control

250

500

1000

Control

250

500

1000

No. of offsprings

13

12

8

13

10

13

16

9

Final body weight (g)

893.0 ± 45.3

1021.2 ± 45.4 **

931.9 ± 55.5

957.3 ± 52.4

1005.5 ± 53.7

1093.1 ± 46.6

860.0 ± 31.6 *

1071.7 ± 58.1

Liver (g)

36.49 ± 2.52

45.08 ± 1.52 *

38.67 ± 3.10

34.91 ± 2.76

41.79 ± 3.39

13.95 ± 3.18

34.68 ± 1.90

48.30 ± 6.14

Lungs (g)

5.53 ± 0.35

6.25 ± 0.26

5.98 ± 0.36

5.46 ± 0.18

5.91 ± 0.25

5.94 ± 0.28

5.72 ± 0.18

5.93 ± 0.45

Heart (g)

2.63 ± 0.17

3.50 ± 0.16 **

2.86 ± 0.17

3.04 ± 0.19

3.19 ± 0.17

3.38 ± 0.21

2.72 ± 0.12 *

3.34 ± 0.20

Spleen (g)

0.71 ± 0.05

0.77 ± 0.04

0.71 ± 0.08

0.81 ± 0.04

0.64 ± 0.06

0.74 ± 0.05

0.74 ± 0.04

0.78 ± 0.07

Thymus (g)

2.31 ± 0.20

2.51 ± 0.23

2.11 ± 0.38

1.96 ± 0.11

2.40 ± 0.30

2.77 ± 0.19

1.671.14 *

2.36 ± 0.31

Kidneys (g)

7.67 ± 0.42

9.80 ± 0.46 **

8.29 ± 0.29

8.58 ± 0.52

8.82 ± 0.46

8.40 ± 0.30

7.86 ± 0.37

9.56 ± 0.55

Thyroids (mg)

75.89 ± 8.35

102.68 ± 4.18*

86.74 ± 10.97

80.93 ± 7.41

82.78 ± 8.00

89.90 ± 4.11

75.75 ± 5.43

96.30 ± 9.67

Adrenals (mg)

69.65 ± 6.02

9.1.93 ± 1.06 **

78.79 ± 5.89

71.36 ± 6.00

82.23 ± 4.37

94.24 ± 5.07

87.64 ± 4.18

105.39 ± 15.11

Testes or Ovaries

(mg)

180.42 ± 17.15

272.86 ± 16.46 **

185.62 ± 23.78

162.84 ± 20.59

46.31 ± 7.90

46.10 ± 2.80

43.53 ± 2.69

47.77 ± 3.59

* Significantly different from the control at 5% level

** Significantly different from the control at 1% level

Table 4: Relative organ weights per 100 g of rabbit offsprings

 

Male

Female

 

Control

250

500

1000

Control

250

500

1000

No. of offsprings

13

12

8

13

10

13

16

9

Final body weight (g)

893.0 ± 45.3

1021.2 ± 45.4 **

931.9 ± 55.5

957.3 ± 52.4

1005.5 ± 53.7

1093.1 ± 46.6

860.0 ± 31.6 *

1071.7 ± 58.1

Liver (g/100 g)

4.08 ± 0.25

3.99 ± 0.22 *

4.16 ± 0.26

3.52 ± 0.10

4.25 ± 0.17

4.03 ± 0.21

4.02 ± 0.16

4.04 ± 0.32

Lungs (g/100 g)

0.61 ± 0.03

0.55 ± 0.03

0.65 ± 0.04

0.58 ± 0.03

0.62 ± 0.02

0.55 ± 0.02 *

0.67 ± 0.03

0.51 ± 0.02 **

Heart (g/100 g)

0.29 ± 0.01

0.31 ± 0.01

0.31 ± 0.01

0.31 ± 0.02

0.33 ± 0.01

0.31 ± 0.01

0.32 ± 0.01

0.29 ± 0.01 *

Spleen (g/100 g)

0.08 ± 0.01

0.07 ± 0.01

0.08 ± 0.01

0.08 ± 0

0.07 ± 0.01

0.07 ± 0.01

0.09 ± 0

0.07 ± 0.01

Thymus (g/100 g)

0.25 ± 0.02

0.22 ± 0.02

0.25 ± 0.03

0.20 ± 0.01 *

0.24 ± 0.03

0.25 ± 0.01

0.195 ± 0.01

0.20 ± 0.02

Kidneys (g/100 g)

0.85 ± 0.04

0.86 ± 0.04

0.90 ± 0.03

0.88 ± 0.02

0.91 ± 0.02

0.80 ± 0.01

0.91 ± 0.01

0.83 ± 0.03 *

Thyroids (mg/100 g)

8.21 ± 0.69

9.10 ± 0.51

9.21 ± 0.87

8.18 ± 0.42

8.40 ± 0.56

8.41 ± 0.45

8.81 ± 0.57

8.13 ± 0.41

Adrenals (mg/100 g)

7.82 ± 0.60

8.37 ± 0.40

8.58 ± 0.77

7.18 ± 0.28

8.61 ± 0.51

8.79 ± 0.57

9.15 ± 0.42

9.04 ± 0.14

Testes or Ovaries

(mg/100 g)

23.68 ± 1.31

19.68 ± 0.94 *

19.48 ± 1.69

16.35 ± 1.56

5.15 ± 1.19

4.33 ± 0.32

5.19 ± 0.43

3.84 ± 0.32

* Significantly different from the control at 5% level

** Significantly different from the control at 1% level

Table 5: Effects of d-limonene on gross differentiations of rabbit offsprings

 

Control

250

500

1000

No. of examined offsprings

23

25

24

22

Days of gross differentiation after birth

Opening of the ear-shell

 

 

 

 

6th day (%)

0

0

1 (4.2)

0

7th day (%)

23 (100)

25 (100)

23 (95.8)

22 (100)

Coating with the hair

 

2nd day (%)

7 (30.4)

0

0

0

3rd day (%)

16 (69.6)

25 (100)

24 (100)

22 (100)

Odontiasis

 

At birth (%)

23 (100)

25 (100)

24 (100)

22 (100)

Opening of the eyelids

 

9th day (%)

0

0

0

3 (13.6)

10th day (%)

11 (47.8)

4 (16.0)

13 (54.2)

5 (22.7)

11th day (%)

4 (17.4)

15 (60.0)

10 (41.7)

12 (54.5)

12th day (%)

3 (13.0)

5 (20.0)

1 (4.2)

2 (9.1)

13th day (%)

5 (21.7)

1 (4.0)

0

0

Table 6: Effects of d-limonene on postnatal development of rabbit offsprings

 

Control

250

500

1000

No of dams

3

3

3

3

No. of still-birth (Male/Female)

1 (1/0)

0

0

1 (0/1)

No. of offsprings (Male/Female)

At birth

28 (15/13)

27 (14/13)

26 (8/18)

27 (15/12)

1st week

28 (15/13)

27 (14/13)

25 (8/17)

26 (14/12)

2nd week

26 (14/12)

27 (14/13)

25 (8/17)

26 (14/12)

3rd week

24 (14/10)

27 (14/13)

25 (8/17)

25 (14/11)

4th week

23 (13/10)

26 (13/13)

25 (8/17)

22 (13/ 9)

5th week

23 (13/10)

25 (12/13)

25 (8/17)

22 (13/ 9)

6th week

23 (13/10)

25 (12/13)

25 (8/17)

22 (13/ 9)

7th week

23 (13/10)

25 (12/13)

24 (8/16)

22 (13/ 9)

Weanling rate (%)

79.3 (81.2/76.9)

92.6 (85.7/100)

92.3 (100/88.9)

78.6 (86.7/69.2)

Table 7: Postnatal examinations of rabbit offsprings

 

Control

250

500

1000

No. of dams

3

3

3

3

No. of examined offsprings

23

25

24

22

Sensory function

Normal

Normal

Normal

Normal

External examination

No. of malformed offsprings

0

0

0

0

Visceral examination

No. of malformed offsprings

0

0

0

0

No. of minor abnormality

Incomplete lobulation of lungs (%)

2 (8.7)

1 (4.0)

0

0

Accessory spleen (%)

2 (8.7)

0

0

0

Protrusion of gall bladder (%)

1 (4.3)

1 (4.0)

0

0

Skeletal examination

No. of malformed offsprings

0

0

0

0

No. of variation

Left lumbar rib (%)

4 (17.4)

4 (16.0)

4 (16.7)

4 (18.2)

Right lumbar rib (%)

2 (8.7)

6 (24.0)

6 (25.0)

4 (18.2)

Translocation of caudal vertebrae (%)

1 (4.3)

0

1 (4.2)

0

Ossification pattern

Retarded ossification of 5th sternebrae (%)

0

2 (8.0)

0

1 (4.5)

Accessory ossification center of 5th sternebrae (%)

1 (4.3)

2 (8.0)

0

3 (13.6)

Conclusions:
Under the test conditions, d-limonene was not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity was considered to be greater than 1000 mg/kg bw/day. The NOAEL for maternal toxicity was considered to be 250 mg/kg bw/day based on the decreased bodyweight gain.
Executive summary:

In a prenatal developmental toxicity study, d-limonene was administered orally to groups of pregnant Japanese white rabbits at dose levels of 250, 500 and 1000 mg/kg bw/day for 13 days from Day 6 to 18 of gestation. Food consumption and bodyweights of pregnant rabbits were recorded during organogenesis. Caesarean sections were performed and the number of dead, live or resorbed fetuses, sex ratio and number of implantation sites were recorded. Fetuses were weighed and examined for external, visceral and skeletal malformations. Treatment with the highest dose level (1000 mg/kg bw/day) of d-limonene resulted in death of 6/18 dams (33% mortality). The significant decrease of bodyweight gain and food consumption were temporarily observed in dams given 500 and 1000 mg/kg bw/day of d-limonene, but no anomalies were observed in the general behavior of dams given 250 and 500 mg/kg bw/day of d-limonene during the gestation. External examination of fetuses showed no anomalies. Visceral and skeletal examinations revealed some anomalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae. These did not appear to be dose-dependent and restored to normal during the postnatal development. Other non specific anomalies involved the lumber ribs in fetuses and offsprings, formation of the accessory ossification center of the 5th sternebrae in offsprings and the atrial septal defect detected in only 2 fetuses of a litter from dams treated with 250 mg/kg bw/day of d-limonene. Under the test conditions, d-limonene was not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity was considered to be greater than 1000 mg/kg bw/day. The NOAEL for maternal toxicity was considered to be 250 mg/kg bw/day based on the decreased bodyweight gain.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
21 April 1992 – 05 August 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Test method according to OECD guideline 414 and GLP, but only two dose levels were tested.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(Only two dose levels were tested)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Lippische Versuchstierzucht, HAGEMANN GmbH, D-4923 Extertal 1, Stock: Tif: RAI f (SPF)
- Age at study initiation: ca. 54 days old
- Weight at study initiation: 180 - 190 g
- Housing: The dams were kept singly in MAKROLON cages type III.
- Diet (e.g. ad libitum): ALTROMIN 1314 (supplied by: ALTROMIN GmbH, P.O.Box 285, D-4937 Lage/Lippe) served as food, ad libitum.
- Water (e.g. ad libitum): Tap water, ad libitum.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 ºC
- Humidity (%): 50 ± 15 %
- Photoperiod: 12 hrs dark / 12 hrs light (150 lux at 1.50 m room height)

IN-LIFE DATES: 28 April 1992 – 05 August 1992

Route of administration:
oral: gavage
Vehicle:
other: sesame oil, DAB 10
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance-vehicle mixtures were freshly prepared each day immediately before dosing.
The stability and homogeneity of the solution was ensured for a period of 4 hours

VEHICLE
- Concentration in vehicle: 50 mg/ml and 200 mg/ml
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the determination of the concentration in the substance-vehicle preparations, 2 samples of 10 mL (each one for the concentrations 50.0 mg/Camphene/mL suspension and 200.0 mg Camphene/mL suspension) were analyzed at the start and at the end of treatment.
The determination of the concentrations was performed by GC using a FID detector.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: Females randomly chosen were mated overnight with fertile ('proved') 4 - 12 months old male rats of the same breed. They were repeatedly employed, at the earliest three days after successful copulation.
- M/F ratio per cage: 1 male/1 female
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From the 6th to 15th day of pregnancy
Frequency of treatment:
Daily
Duration of test:
20 days (from day 0 to day 20 of pregnancy)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 pregnant female rats per group (plus 15 reserve animals)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
During a range-finding study using 3 pregnant rats from the same strain as selected for the present study, a camphene dose-Ievel of 1000 mg/kg b.w., by gavage, was administered from day 6 to 15 of pregnancy. 1000 mg/kg b.w. is the highest requested dose according to the OECD method 414 (limit test for embryotoxicity).
Except for a slight transient reaction after the first dosing, 1000 mg/kg b.w. was well-tolerated by the dams. 1000 mg/kg b.w. did not influence the prenatal development. Based on these results, 250 mg and 1000 mg camphene/kg b.w./day, by gavage, treatment from the 6th to 15th day of prenancy were selected by the sponsor as doses for the main experiment.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily checks (once in the morning and once as late on the day as practicable) were performed on behaviour, external appearance, mortality and faeces.

BODY WEIGHT: Yes
- Time schedule for examinations: daily (always at the same time in the morning)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption was determined daily by weighing the residue.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Intake of drinking-water was observed daily

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: On the 20th day of gestation the surviving rats were laparotomised under ether narcosis. The ovaries and uteri were removed and examined. To check for possible drug effects a dissection was carried out which included macroscopic examination of internal organs. A full macroscopic inspection was carried out in the prematurely deceased dams as soon as possible after their exitus.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes. The uterus weight was determined (with and without fetuses), weight of the ovaries was determined.
- Number of corpora lutea: Yes.
- Number of implantations: Yes. Implantations and location of fetuses in the uterus were determined.
- Number of early resorptions: Yes.
- Number of late resorptions: Yes.
- Other:
Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
Weights and length of fetuses and weight of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]. According to the method of Wilson.
- Skeletal examinations: Yes: [half per litter]. According to the method of Dawson.
Statistics:
The comparison of malformation and variation rates was carried out using R. A. FISHER's exact test (p ≤ 0.05). All other data were evaluated in the following way:
Homogeneity of variances was tested by the Bartlett chi-square test, and, if the variances were homogeneous, a one-way analysis of variance was applied. When the results indicated a significant difference among groups the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Six of the 20 dams treated with 1000 mg/kg bw/day showed reduced motor activity and salivation after first dosing, one of them salivation after second dosing. One more dam showed salivation on the second dosing. The reactions occurred within 5 - 20 min after administration and lasted for 20 - 60 min, 1-2 hrs or 2-6 hrs.
No clinical signs were observed in the remaining high-dosed and the low-dosed dams.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two dams treated with 1000 mg/kg bw/day died prematurely probably due to regurgitation and aspiration of part of the administered test compound. Because deaths were no substance-related these dams were replaced to have 20 pregnant females in this dose group for study examinations.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gain remained within the normal range of the controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Transient impairment of the food consumption by the highest tested dose (1000 mg/kg bw/day) was observed on the 7th, 8th and 9th gestation day by 6%, 22% and 10%, respectively.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Treatment did not influence drinking-water consumption.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Six of the 20 dams treated with 1000 mg/kg bw/day showed reduced motor activity and salivation after first dosing, one of them salivation after second dosing. One more dam showed salivation on the second dosing. The reactions occurred within 5 - 20 min after administration and lasted for 20 - 60 min, 1-2 hrs or 2-6 hrs.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The uterus weights of the treated groups were not influenced by the exposure to the test compound.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No substance-related pathological changes were detected at autopsy.
Macroscopic inspection during dissection of the two deceased rats revealed emphysematous and spongy lungs, these findings indicating regurgitation and aspiration of part of the administered test compound.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were registered either in treated groups or in the control group.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
There were no substance-related and/or statistically significant differences between the treated groups and the control group for the pre-implantation loss.
However, the high dose caused a slight but not significant (at p < = 0.01) increase of the post-implantation loss (substance-treated group: 11.5%, control group: 5.2%).
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
The mean value of total losses by resorptions per litter were calculated to be 0.8 in the control group, 1.0 in the low dose group and 1.3 in the high dose group (statistically not significant at p < = 0.01).
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
1000 mg camphene/kg b.w./day caused a slight but not significant (at p < = 0.01) increase of the resorption rate (substance-treated group: 11.5%, control group: 5.2%).
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses occurred in the substance treated and control dams.
The treated groups had a number of viable fetuses per pregnant rat similar to the control group.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were no substance-related and/or statistically significant differences between the treated groups and the control group in conception rate, in the mean number of corpora lutea and implantation site.
The conception rate varied between 84% and 96%.
Other effects:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights and mean placental weights were not influenced by the test substance exposure. All values were within the range of biological variation.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no substance-related and/or statistically significant differences between the treated groups and the control group in the number of viable fetuses.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in the treated groups was comparable with the control fetuses. The differences observed in comparison to the control are without any biological relevance.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
External examination of the fetuses revealed malformations (shifted and fused dorsal, lumbar and coccygeal vertebrae, bilateral crossed legs, stump tail, omphalocele) in one fetus out of 243 fetuses in the high dose group and only one variation (haemorrhage in the left anterior chamber of the eye) in one fetus out of 248 fetuses in the low dose group. One out of 258 control foetuses showed one malformation (stump tail). These changes belong to the spontaneous range as to their type and the number of affected foetuses.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No malformations were observed during skeletal examinations.
The variations elicited were related to the ribs (accessory 14th ribs, wavy ribs) and the sternum (sternebra bipartite or misaligned) and were found in all groups, including the control, to about the same extent.
In all groups signs of retardations (incomplete or missing ossification of hyoid, skull, vertebral bodies and/or sternebra were found without any clear differences of biological relevance between the groups, included the control).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The examination of the organ of the foetuses revealed no malformations in the treated groups.
Variations were detected in all groups. The very common findings (uni- or bilateral dilated renal pelvis, haemorrhages of the liver, 4th cerebral ventricle enlarged) in the rat strain used in the study occurred without any dose-response relationship.
The type and number of variations found in the treated groups were similar to those found in the control group.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Fetuses toxicity
Remarks on result:
other: No adverse effect observed at the highest dose tested
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
other: (shifted and fused dorsal, lumbar and coccygeal vertebrae, bilateral crossed legs, stump tail, omphalocele)
Description (incidence and severity):
One malformed fetus at 1000 mg/kg bw/day belongs to the spontaneous range as to type and number of affected fetuses (control: one fetus with stump tail).
Key result
Developmental effects observed:
no
Conclusions:
Under the test conditions, the NOEL for the dams and for the fetal organism was 250 mg camphene/kg bw/day. Camphene did not possess teratogenic properties.
Executive summary:

A pre-natal developmental toxicity test was performed with camphene according to OECD Guideline 414. 20 pregnant Sprague-Dawley rats were orally exposed by gavage during the 'critical' phase of organogenesis (daily from the 6th to 15th day of pregnancy). Tested concentrations were 0, 250 and 1000 mg/kg bw/day, using sesame oil as vehicle. Their development during the gestation period was observed. On day 20 of pregnancy the animals were laparotomised and examined macroscopically for implantation sites, resorptions in the uterus and for condition of the fetuses.

 No substance-related mortality was observed in the dams. Six of the 20 dams treated with 1000 mg/kg bw/day showed reduced motor activity and salivation. No other clinical signs were observed in the remaining high-dosed and the low-dosed dams. Body weights remained within the normal range and body weight gain showed no influence of the test compound. Transient impairment of the food consumption by the highest tested dose was observed. Treatment did not influence drinking-water consumption. No substance-related pathological changes were detected at autopsy.

The highest tested dose caused a slight but not significant (at p<= 0.01) increase of the resorption rate and, consequently, of the post-implantation loss. No further influence on the prenatal development was detected. All other fetal parameters were within the normal range of the control group. External macroscopic inspection, examination of soft tissue (WILSON's method) and skeletal examination (staining of the skeleton according to DAWSON's method) revealed no substance-related variations and/or retardations. One malformed fetus at 1000 mg/kg bw/day (shifted and fused dorsal, lumbar and coccygeal vertebrae, bilateral crossed legs, stump tail, omphalocele) belongs to the spontaneous range as to type and number of affected fetuses (control: one fetus with stump tail). No dead fetuses occurred in the substance treated and control dams.

Under these test conditions, the NOEL for the dams and for the fetal organism was 250 mg camphene/kg bw/day. Camphene did not possess teratogenic properties.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
03 January 1992 – 02 April 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Dose-Range-Finding study. Test method according to OECD guideline 414 and GLP.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(Only three animals were used per group)
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Lippische Versuchstierzucht, HAGEMANN GmbH, D-4923 Extertal 1
- Age at study initiation: ca. 71 days old
- Weight at study initiation: 211 - 245 g
- Housing: The dams were kept singly in MAKROLON cages type III.
- Diet (e.g. ad libitum): ALTROMIN 1314 (supplied by: ALTROMIN GmbH, P.O.Box 285, D-4937 Lage/Lippe) served as food, ad libitum.
- Water (e.g. ad libitum): Tap water, ad libitum.
- Acclimation period: Five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 ºC
- Humidity (%): 50 ± 15 %
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 03.01.1992 To: 23.01.1992
Route of administration:
oral: gavage
Vehicle:
other: sesame oil, DAB 9
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance-vehicle mixtures were freshly prepared each day immediately before dosing.
Volume of administration: 5 mL/kg bw
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Fertile ('proved') 4 - 12 months old male rats of the same breed served as partners. They were repeatedly employed, at the earliest three days after successful copulation. The female breeding partners were randomly chosen.
Matings were monogamous.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From the 6th to 15th day of pregnancy
Frequency of treatment:
Daily
Duration of test:
20 days (from day 0 to day 20 of pregnancy)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
3 pregnant female rats per group (plus 2 reserve animals)
Control animals:
yes, concurrent vehicle
Maternal examinations:
Daily checks (once in the morning and once as late on the day as practicable) were performed on behaviour, external appearance, mortality and faeces.
Body weight was ascertained daily - always at the same time in the morning - and these measurements were also used for calculating the daily amount of test compound to be administered.
Food consumption was determined daily by weighing the residue. Intake of drinking-water was observed daily.
Ovaries and uterine content:
On the 20th day of gestation the surviving rats were laparotomised under ether narcosis. The ovaries and uteri were removed and examined. To check for possible drug effects a dissection was carried out which included macroscopic examination of internal organs.
Fetuses were removed and the following examinations performed:
A count was taken of fetuses and placentae.
Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
Number and size of resorptions were determined.
Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
The uterus weight was determined (with and without fetuses), weight of the ovaries was determined.
Weights and length of fetuses and weight of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the
mean litter weight).
Fetal examinations:
Fetuses were inspected externally for damages, especially for malformations.
Fetuses were dissected and the number and type of possible variations (incl. retardations) or malformations was determined macroscopically.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Two of the three dams treated with 1000 mg Camphene/kg bw showed reduced motor activity and salivation after the first dosing. These reactions occurred within 20 minutes after gavage treatment and lasted for 1-2 hours in dam nº. 5 and for 2-6 hours in dam nº. 6. No corresponding clinical signs were seen at the further observations and examinations, the third dam used remained inconspicuous. Consistency of faeces did not differ between substance-treated and control rats.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
None of the dams died prematurely.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
1000 mg Camphene/kg bw/day had no influence on body weight gain of the dams. All values remained within the range of the controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
1000 mg Camphene/kg bw/day had no influence on food consumption of the dams. All values remained within the range of the controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
1000 mg Camphene/kg bw/day had no influence on drinking-water consumption of the dams. All values remained within the range of the controls.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Two of the three dams treated with 1000 mg Camphene/kg bw showed reduced motor activity and salivation after the first dosing. These reactions occurred within 20 minutes after gavage treatment and lasted for 1-2 hours in dam nº. 5 and for 2-6 hours in dam nº. 6.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The uterus and ovaries weights of the treated groups were not influenced by the exposure to the test compound.
Gross pathological findings:
no effects observed
Description (incidence and severity):
None of the dams showed macroscopically visible organic changes at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Number of implantations showed no abnormalities.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Resorptions were within the normal range.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Resorptions were within the normal range.
Dead fetuses:
no effects observed
Description (incidence and severity):
Macroscopic inspection during laparotomy revealed that dead foetuses did not occur.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Number of corpora lutea, implantations and foetuses showed no abnormalities.
Other effects:
no effects observed
Dose descriptor:
NOAEL
Effect level:
< 1 000 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Foetal weights and length as well as weight of the placentae did not differ substance-relatedly from the controls.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Description (incidence and severity):
Macroscopic inspection during laparotomy revealed no variations/retardations or malformed foetuses. None of the foetuses was a runt.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Macroscopic inspection during laparotomy revealed no variations/retardations or malformed foetuses. None of the foetuses was a runt.
Visceral malformations:
no effects observed
Description (incidence and severity):
Macroscopic inspection during laparotomy revealed no variations/retardations or malformed foetuses. None of the foetuses was a runt.
Other effects:
no effects observed
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
Under the present test conditions, Camphene did not influence the prenatal development. Except for a slight transient reaction after the first dosing (reduced motor activity, salivation) the dose-level of 1000 mg/kg bw/day was well-tolerated by the dams.
Executive summary:

Pregnant Sprague-Dawley rats were orally treated with the test substance by gavage during the 'critical' phase of organogenesis (daily from the 6th to 15th day of pregnancy). Tested concentrations were 0 and 1000 mg/kg bw/day, using sesame oil as vehicle. 3 pregnant rats were used in each group. Their development during the gestation period is observed. On day 20 of pregnancy the animals were laparotomised and examined macroscopically for implantation sites, resorptions in the uterus and for condition of the fetuses.

Influence on the dam:

None of the dams died prematurely. Two of the three treated dams showed reduced motor activity and salivation after the first dosing. These reactions occurred within 20 minutes after gavage treatment and lasted for 1-2 hours in dam nº. 5 and for 2-6 hours in dam nº. 6. No corresponding clinical signs were seen at the further observations and examinations, the third dam used remained inconspicuous. Consistency of faeces did not differ between substance-treated and control rats. Test substance had no influence on body weight gain, food and drinking-water consumption of the dams. All values remained within the range of the controls. None of the dams showed macroscopically visible organic changes at necropsy.

Influence on the fetus:

Camphene did not influence the prenatal development of rats. Number of corpora lutea, implantations and foetuses showed no abnormalities, the resorptions were within the normal range. Foetal weights and length as well as weight of the placentae did not differ substance-relatedly from the controls. Macroscopic inspection during laparotomy revealed no variations/retardations, malformed or dead foetuses did not occur. None of the foetuses was a runt.

Under the present test conditions, Camphene did not influence the prenatal development at 1000 mg/kg bw/day. Except for a slight transient reaction after the first dosing (reduced motor activity, salivation) this dose-level was well-tolerated by the dams.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Prenatal developmental toxicity study: Groups of pregnant Wistar rats (20/dose: 15 for teratogenicity study, 5 for postnatal development) were administered orally with d-limonene at dose levels of 0, 591 and 2869 mg/kg bw/day suspended with 1% gum-arabic solution for 7 days from Day 9 to 15 of gestation and evaluated for developmental toxicity.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
No data
Route of administration:
oral: unspecified
Vehicle:
other: 1% gum-arabic solution
Details on exposure:
Volume administered: 5 mL/kg bw for all doses
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
None
Details on mating procedure:
no data
Duration of treatment / exposure:
7 days (gestation Day 9-15)
Frequency of treatment:
Once daily
Duration of test:
Gestation Day 0 to postnatal week 7
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
591 mg/kg bw/day
Dose / conc.:
2 869 mg/kg bw/day (nominal)
No. of animals per sex per dose:
20 pregnant rats
Control animals:
yes, concurrent vehicle
Details on study design:
No data
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data
General behaviour observed, but no data regarding the frequency of observation

BODY WEIGHT: Yes
- Time schedule for examinations: daily

POST-MORTEM EXAMINATIONS: No data
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: No data
Examinations included: Number of implantations, number of resorptions and foetus bodyweight and placental weight
Fetal examinations:
- External examinations: Yes
- Visceral examinations: Yes
- Skeletal examinations: Yes
Statistics:
statistical significance difference of effects from controls were calculated at 5% level.
Indices:
No data
Historical control data:
No data
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Deaths (40%) and decreased bodyweight gain at 2869 mg/kg bw/day
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Deaths (40%) and decreased bodyweight gain at 2869 mg/kg bw/day
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
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:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
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
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
591 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Delayed ossification of fetuses metacarpal bone and proximal phalanx at 2869 mg/kg bw/day was caused significantly, compared with the control group, but this was restored to normal within several weeks after birth.
Visceral malformations:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
A tendency of decreased bodyweight was noted in postnatal male offsprings born to mothers treated at 2869 mg/kg bw/day, compared with the control group.
Thymus, spleen and ovaries weights decreased in offsprings born to mothers treated at 2869 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
591 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
other: Decreased bodyweight gain (male offsprings) and organ weights at 2869 mg/kg bw/day
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: skeletal: metacarpals and proximal phalanges
Description (incidence and severity):
Prolongation of the ossification of metacarpals and proximal phalanges
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
2 869 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
no
Relevant for humans:
not specified

Table 1: Body weight changes in pregnant rats treated orally with d-limonene

Dose

(mg/kg bw)

Gestational days

Gain

0

9

12

16

20

Control

214.80 ± 32.55

248.70 ± 28.92

265.60 ± 30.53

289.85 ± 35.01

325.30 ± 44.01

105.50 ± 29.67

591

221.25 ± 39.58

254.65 ± 41.16

264.80 ± 39.94

290.10 ± 38.37

325.60 ± 52.75

103.95 ± 19.38

2869

214.75 ± 4.57

258.75 ± 32.76

 

248.92 ± 26.27

263.17 ± 22.96 *

305.00 ± 27.07

90.25 ± 22.38

* Significantly different from the control, P <0.05

Table 2: Effects of d-limonene on rat fetuses

Dose (mg/kg bw)

Control

591

2869

No. of mothers

15

15

15

Mortality of mothers (%)

0

0

40

No. of total implants

12.73 ± 2.96

12.18 ± 3.65

10.44 ± 3.71

No. of dead fetuses

0

0

0

No. of resorbed fetuses

1.00 ± 1.10

1.47 ± 2.42

0.89 ± 0.73

No. of live fetuses

176

162

87

Sex ratio (Male/Female)

0.69

1.22

0.85

Fetuses body weight (g)

Male

3.71 ± 0.45

3.53 ± 0.35

3.73 ± 0.52

Female

3.46 ± 0.44

3.38 ± 0.45

3.63 ± 0.40

Placental weight(g)

Male

0.49 ± 0.07

0.49 ± 0.10

0.48 ± 0.06

Female

0.47 ± 0.07

0.46 ± 0.06

0.44 ± 0.05

Malformation

External

0

0

0

Visceral

1

0

0

Table 3: Effects of d-limonene on skeletal development of rat fetuses

Dose (mg/kg bw)

Control

591

2869

No. of examined fetuses

83

84

42

Variation

Shortness of 13th rid

1

0

0

Lumbar rid

0

1

2

Asymmetry of sternebrae

0

0

1

Ossification

Delayed ossification of parietal bone

2

0

0

Non-ossification of occipital bone

 

0

4

1

Non-ossification of parietal bone

0

3

0

No. of ossified metacarpal bone

7.69 ± 0.72

7.49 ± 0.84

6.97 ± 0.96 *

No. of ossified proximal phalanx (Forelimb)

2.48 ± 1.71

2.25 ± 1.81

0.55 ± 1.28 *

No. of ossified metatarsal bone

 

7.98 ± 0.56

8.01 ± 011

8.00 ± 0

No. of ossified

sternebraea

5.47 ± 0.98

5.60 ± 0.71

5.52 ± 0.73

No. of ossified

caudal vertebrae

3.76 ± 0.64

3.80 ± 0.57

3.95 ± 0.68

* Significantly different from the control, P <0.05

Table 4: Body weight changes of postnatal rat offsprings born to mothers treated orally with d-limonene

Postnatal

weeks

Males

Females

Dose (mg/kg bw)

Dose (mg/kg bw)

Control

591

2869

Control

591

2869

0

5.19 ± 0.55

5.46 ± 0.52

4.79 ± 0.46

4.93 ± 0.62

5.09 ± 0.68

4.89 ± 0.66

1

13.06 ± 1.50

12.49 ± 0.99

10.62 ± 1.54 *

12.82 ± 1.59

12.22 ± 1.07

10.66 ± 1.84

2

26.06 ± 3.12

24.86 ± 2.74

22.67 ± 5.05 *

25.88 ± 3.35

24.31 ± 2.42

22.72 ±.3.92

3

41.91 ± 5.89

39.55 ± 5.14

40.77 ± 5.16

41.08 ± 5.59

38.56 ± 4.37

38.39 ± 4.96

4

73.12 ± 9.89

71.33 ± 8.87

67.67 ± 8.02

69.66 ± 9.43

67.38 ± 6.71

66.01 ± 9.29

5

122.32 ± 12.25

116.47 ± 12.78

112.77 ± 12.65 *

109.57 ± 10.61

107.58 ± 7.95

107.10 ± 13.34

6

176.04 ± 15.80

164.68 ± 16.69

163.11 ± 17 .85 *

141.39 ± 10.54

140.11 ± 9.40

139.45 ± 14.22

7

235.52 ± 17.72

222.43 ± 18.57

213.64 ± 20.10 *

173.06 ± 8.89

169.65 ± 11.13

167.84 ± 15.86

* Significantly different from the control, P <0.05

Table 5: Effects of d-limonene on postnatal development of the rats

Dose

(mg/ kg bw)

Days of postnatal development

Opening of the ear-shell

Coating with
the hair

Odontiasis

Opening of the eyelid

Descending of the testis

Opening of the vaginal orifice

Control

2.55 ± 0.76

5.51 ± 0.91

10.1 ± 0.96

14.83 ± 0.55

22.5 ± 1.30

35.6 ± 2.50

591

2.09 ± 0.82

6.00 ± 0

10.4 ± 0.71

15.00 ± 0.76

21.6 ± 1.39

35.5 ± 1.75

2869

2.41 ± 0.49

8.50 ±0.50

10.4 ± 1.85

15.14 ± 0.75

21.27 ± 0.57

35.93 ± 2.20

Table 6: Effects of d-limonene on development of rat offsprings

Dose (mg/kg)

Control

591

2869

No. of mothers

5

5

5

Mortality of mothers

0

0

40

No. of offspring from birth

to the 7th week

0

61

65

33

1

53

63

30

2

53

63

30

3

53

63

28

4

53

63

28

5

53

63

28

6

53

63

28

7

53

63

28

External abnormality

0

0

0

No. of total implants

13.6 ± 3.1

14.8 ± 1.7

13.7 ± 1.7

No. of dead fetuses at birth

4

4

5

Parturient rate

95

92

93

Weaning rate

89

97

85

Table 7: Absolute organ weights of postnatal rat offsprings born to mothers treated orally with d-limonene

Sex

Dose

(mg/kg bw)

No. of

offsprings.

Final BW (g)

Pituitary (mg)

Thyroids (mg)

Thymus (g)

Lungs (g)

Heart (g)

Spleen (g)

Kidneys (g)

Liver (g)

Adrenals (g)

Testes (g) or Ovaries (mg)

Male

Control

27

235.5 ± 17.7

10.04 ± 1.95

14.06 ± 2.52

0.77 ± 0.08

1.15 ± 0.10

0.82 ± 0.08

0.75 ± 0.11

2.18 ± 0.36

11.55 ± 1.14

38.85 ± 7.37

2.15 ± 0.17

591

29

222.4 ± 18.6

9.58 ± 4.84

13.12 ± 2.77

0.72 ± 0.09

1.10 ± 0.11

0.81 ± 0.08

0.69 ± 0.08

2.10 ± 0.20

11.24 ± 1 .49

36.77 ± 6.93

2.13 ± 0.28

2869

11

213.6 ± 20.1

9.69 ± 0.65

14.41 ± 3.60

0.66 ± 0.08 *

1.19 ± 0.17

0.80 ± 0.07

0.63 ± 0.07 *

2.23 ± 0.38

11.64 ± 1.64

43.23 ± 10.91

2.18 ± 0.14

Female

Control

25

173.1 ± 8.9

11.18 ± 3.15

12.26 ± 1.32

0.59 ± 0.08

0.97 ± 0.11

0.66 ± 0.07

0.52 ± 0.06

1.72 ± 0.22

8.88 ± 0.85

45.51 ± 8.01

77.24 ± 22.01

591

34

169.7 ± 11.7

10.05 ± 3.34

11.57 ± 1.62

0.54 ± 0.07

0.96 ± 0.07

0.67 ± 0.05

0.50 ± 0.06

1.60 ± 0.15 *

8.16 ± 0.85

46.56 ± 8.45

85.84 ± 42.52 *

2869

17

167.8 ± 15.9

9.95 ± 1.87

12.31 ± 1.80

0.51 ± 0.07 *

0.94 ± 0.10

0.62 ± 0.06

0.44 ± 0.04 *

1.62 ± 0.17 *

8.50 ± 0.58

47.15 ± 6.63

63.15 ± 7.99

* Significantly different from the control, P <0.05

Table 8: Relative organ weights per 100 g body weights of postnatal rat offsprings born to mothers treated orally with d-limonene

Sex

Dose

(mg/kg bw)

No. of

offsprings.

Final BW (g)

Pituitary (mg/100 g)

Thyroids (mg/100 g)

Thymus (mg/100 g)

Lungs (mg/100 g)

Heart (mg/100 g)

Spleen (mg/100 g)

Kidneys (g/100 g)

Liver (g/100 g)

Adrenals (mg/100 g)

Testes or Ovaries (mg/100 g)

Male

Control

27

235.5 ± 17.7

4.31 ± 0.78

5.98 ± 0.99

0.33 ± 0.04

0.48 ± 0.04

0.36 ± 0.03

0.31 ± 0.05

0.93 ± 0.08

4.89 ± 0.36

16.49 ± 2.71

0.90 ± 0.04

591

29

222.4 ± 18.6

4.02 ± 0.50

5.93 ± 0.86

0.33 ± 0.04

0.49 ± 0.04

0.37 ± 0.04

0.33 ± 0.09

0.95 ± 0.07

5.10 ± 0.37

16.39 ± 2.53

0.95 ± 0.08 *

2869

11

213.6 ± 20.1

4.23 ± 0.43

5.93 ± 0.66

0.28 ± 0.03 *

0.51 ± 0.05

0.35 ± 0.02

0.27 ± 0.02 *

0.96 ± 0.06

5.03 ± 0.27

17.23 ± 2.26

0.95 ± 0.09

Female

Control

25

173.1 ± 8.9

6.09 ± 1.08

7.08 ± 0.85

0.34 ± 0.05

0.54 ± 0.04

0.38 ± 0.04

0.30 ± 0.04

0.99 ± 0.12

5.14 ± 0.42

26.38 ± 4.71

47.94 ± 9.78

591

34

169.7 ± 11.7

5.82 ± 0.81

6.85 ± 0.96

0.32 ± 0.04

0.54 ± 0.12

0.40 ± 0.03

0.30 ± 0.03

0.95 ± 0.07

4.83 ± 0.37

27.61 ± 3.76

46.74 ± 10.76

2869

17

167.8 ± 15.9

6.27 ± 1.90

7.31 ± 0.90

0.31 ± 0.03 *

0.57 ± 0.08

0.37 ± 0.03

0.27 ± 0.04 *

0.94 ± 0.06

5.14 ± 0.33

26.75 ± 2.93

36.89 ± 4.25 *

* Significantly different from the control, P <0.05

Conclusions:
Under the test conditions, the NOAEL for maternal toxicity was considered to be 591 mg/kg bw/day based on the deaths and decreased bodyweight gain. The NOAEL for fetal toxicity was considered to be 591 mg/kg bw/day based on the delayed skeletal formation and decreased bodyweight gain.
Executive summary:

In a developmental toxicity study, d-limonene was administered orally to groups of pregnant Wistar rats (20/dose: 15 for teratogenicity study, 5 for postnatal development) at dose levels of 0, 591 and 2869 mg/kg bw/day suspended with 1% gum-arabic solution for 7 days from Day 9 to 15 of gestation. Bodyweight of pregnant rats were recorded on Days 0, 9, 12, 16 and 20 during organogenesis. Caesarean sections were performed and the number of dead, live or resorbed fetuses, sex ratio and number of implantation sites were recorded. Fetuses were weighed and examined for external, visceral and skeletal malformations. Number of live offsprings, gross differentiation and organ weights of offsprings were recorded until postnatal week 7.

 

At 2869 mg/kg bw/day, maternal bodyweight decreased and several mothers (40%) died for a period of the treatment, but at 591 mg/kg bw/day, no changes were observed. Delayed ossification of fetuses metacarpal bone and proximal phalanx at 2869 mg/kg bw/day was caused significantly, compared with the control group, but this was restored to normal within several weeks after birth. A decreased tendency of bodyweight was noted in postnatal male offsprings born to mothers treated at 2869 mg/kg bw/day, compared with the control group. Thymus, spleen and ovaries weights decreased in offsprings born to mothers treated at 2869 mg/kg.

 

Under the test conditions, the NOAEL for maternal toxicity was considered to be 591 mg/kg bw/day based on the deaths and decreased bodyweight gain. The NOAEL for fetal toxicity was considered to be 591 mg/kg bw/day based on the delayed skeletal formation and decreased bodyweight gain.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Test method equivalent to OECD guideline 414
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(2 treated groups with fewer than 16 pregnant dams)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Oswaldo Cruz Central Animal House breeding stock.
- Age at study initiation: no data
- Weight at study initiation: 227 ± 22 to 240 ± 23 g
- Housing: animals were housed in standard plastic cages with stainless-steel cover lids and wood shavings as bedding.
- Diet (e.g. ad libitum): pelleted diet (Nuvital ®, Nuvilab Ltd, Curitiba, PR, Brazil), ad libitum.
- Water (e.g. ad libitum): Tap water, ad libitum.
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1ºC
- Humidity (%): 70 %
- Photoperiod: dark/light cycle (lights on from 10.00 hr to 22.00 hr).


Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
(Mazola ®)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
No info.

VEHICLE
- Concentration in vehicle: no info.
- Amount of vehicle (if gavage): 3.75 g/kg bw

Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: Mating was performed by transferring two females to the cage of one male for 2 hr (08.00-10.00 hr).
- M/F ratio per cage: 1 male/2 female
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From the 6th to 15th day of pregnancy
Frequency of treatment:
Daily
Duration of test:
21 days (from day 0 to day 21 of pregnancy)
Dose / conc.:
30 mg/kg bw/day
Dose / conc.:
60 mg/kg bw/day
Dose / conc.:
125 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
No. of animals per sex per dose:
Pregnant female rats per group: 24 (control), 14 (30 mg/kg bw/d), 18 (60 mg/kg bw/d), 25 (125 mg/kg bw/d) and 15 (250 mg(kg bw/d)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Not specified.
Maternal examinations:
BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed on days 0, 6 up to 15 and 21 of pregnancy.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: On day 21 of pregnancy the female rats were anaesthetized with ethyl ether inhalation and killed by decapitation. The gravid uterus was weighed with its contents.

Ovaries and uterine content:
The uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes.
- Number of corpora lutea: Yes.
- Number of implantations: Yes. The number of implantation sites was determined by the method of Salewski (1964).
- Number of resorptions: Yes.
- Other: Sex ratio, weight and viability of fetuses were determined.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [1/3 per litter]. By a microsectioning technique adapted from Sterz (1977). Heart, lungs, thymus, spleen, liver and kidneys of foetuses, which were microdissected, were also weighed.
- Skeletal examinations: Yes: [2/3 per litter]. According to the method of Dawson.

Statistics:
Data were evaluated by one-way analysis of variance or, alternatively, by the Kruskal-Wallis test whenever the data did not fit a normal distribution. Differences between groups were tested by the two-sided Student's t-test or Mann-Whitney U-test. Proportions were analysed by the chi-square test or Fischer's exact test. Statistical evaluation was performed using a MINITAB program (MTB, University of Pennsylvania, 1984), and a difference was considered significant at P < 0.05.
Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no statistically significant difference in pregnancy weight gain between the control and the groups treated with 30 and 60 mg TER/kg body weight but marked reductions in weight gain during the treatment period (days 6-15) were observed in rats exposed to the two highest doses tested (125 and 250mg/kg body weight).
Furthermore, a statistically significant reduction in total pregnancy weight gain minus gravid uterus weight was found at these two highest doses tested (125 and 250mg/kg body weight).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
Treatment did not influence drinking-water consumption.
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 specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
At caesarian section no gross pathological alteration was found in the maternal organs of any group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Neither the number of corpora lutea graviditatis/dam nor the number of visible implantation sites/litter were altered by TER over the dose range tested.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
In any of the TER-treated groups the number of resorptions/litter and the ratio of resorptions/implantation sites were not increased above that of the controls.
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
No alteration in the number of live foetuses/litter was noted in TER treated dams.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
The ratio of pregnant (i.e. rats with implantation sites detected by the method of Salewski at term)/sperm-positive treated dam did not differ significantly from that of the control group in rats treated with doses up to 125 mg TER/kg body weight but it was reduced at 250 mg TER/kg body weight.
Other effects:
no effects observed
Key result
Dose descriptor:
LOEL
Effect level:
125 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
LOEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
changes in number of pregnant
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
A significant reduction in foetal body weight was noted at the highest dose (250mg/kg body weight). No statistically significant reduction in foetal body weight was observed at doses lower than 250 mg/kg body weight.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no substance-related and/or statistically significant differences between the treated groups and the control group in the number of viable fetuses.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in the treated groups was comparable with the control fetuses. The differences observed in comparison to the control are without any biological relevance.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Except for a higher frequency of kinky tail in the group exposed to 30 mg TER/kg body weight and a higher proportion of foetuses with haematoma at the highest dose (250mg/kg body weight), no salient finding was revealed by external examination.
The increased frequency of tail abnormalities (bent tip and kinky tail) observed in foetuses exposed to TER was not related to dose and, in addition, the spontaneous frequency of kinky tail is relatively high in this rat strain (1%).
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Signs of delayed ossification (poorly ossified and not ossified bones as well as irregular spongy bones) were noted at doses higher than 30mg TER/kg body weight.
Also, a dose-related increase in the number of foetuses showing one or more abnormalities was found at doses higher than 30mg TER/kg body weight. These skeletal anomalies were manly higher incidences of os squamosum irregularly shaped, os supraoccipitale incompletely ossified, shorter ribs, extra ribs (cervical), sternum dislocated and os processus deltoid irregularly shaped.

Visceral malformations:
no effects observed
Description (incidence and severity):
No increase in visceral malformations was observed in TER-treated groups.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The reduction in foetal weight noted at the highest dose was accompanied by a decrease in the absolute weights of heart, liver, lungs and thymus. The reduction in thymus weight was particularly pronounced and the relative weight [thymus weight (mg)/foetal weight (g)] of this organ was also decreased. In contrast with the effects of TER on the weight of other foetal organs, the kidneys were heavier in the groups treated with 125 and 250mg TER/kg body weight.
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: skull
skeletal: sternum
skeletal: rib
Description (incidence and severity):
The overall increase in the occurrence of skeletal anomalies seems to result, to a large extent, from higher incidences of os squamosum irregularly shaped, os supraoccipitale incompletely ossified, shorter ribs, extra ribs (cervical), sternum dislocated and os processus deltoid irregularly shaped.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
60 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1: Maternal weight gain of rats treated orally with alpha-terpinene on days 6-15 of pregnancy (a)

 

alpha-terpinene (mg/kg bw/day)

Treatment

0

30

60

125

250

Treated females

28

15

20b

26

27

Pregnant females

24

14

18

25

15

Pregnant/sperm positive females (%)

86

93

90

96

56*

Maternal weight (g)

Day 0

227±20

230±22

229±11

227±18

240±23

Day 21

348±29

347±39

357±23

341±28

324±29*

Gravid uterus weight (g)

71.8±18.1

72.8±23.1

77.0±19.9

76.3±11.0

63.0±18.7

Maternal weight gain (g)

Days 0-6

27.5±8.2

30.8±8.4

31.1±9.0

29.1±7.8

27.3±7.6

Days 6-11

13.6±5.7

16.9±5.7

11.8±5.8

6.3±7.1*

-17.8±12.9*

Days 6-15

30.7±21.9

35.7±9.4

29.2±6.8

21.0±9.1*

1.4±9.7*

Days 15-21

63.0±11.4

64.5±15.2

67.9±12.0

63.9±17.6

55.1±19.3

Days 0-21

121.2±21.9

131.1±23.2

128.3±17.4

114.1±22.1

83.7±27.1*

Days 0-21 (minus uterus weight)

49.4±15.6

58.3±11.5

51.2±14.4

37.7±19.0*

20.7±13.7*

a One pregnant female delivered on day 20.

b Percentage of pregnant females was analysed by the chi-square test. All other parameters were analysed by one-way analysis of variance and Student’s t-test. Values are mean ± SD.

*p < 0.05 v. controls.

Table 2: Parameters assessed at caesarean section of rats treated orally with alpha-terpinene on days 6-15 of pregnancy (a)

 

alpha-terpinene (mg/kg bw/day)

 

0

30

60

125

250

Corpa lutea

12.5±3.0

12.9±2.1

12.6±2.5

12.9±1.8

12.1±2.9

Implantation sites

Total

306

179

232

327

189

Per litter

12.6±3.2

12.8±3.8

12.9±3.1

13.2±1.9

12.6±2.4

Resorptions

Total

37

27

15

27

24

Resorptions/implantations (%)

12.1

15.1

6.5

8.2

12.7

Live foetuses

Total

275

158

218

299

165

Foetuses/implantations (%)

88

85

94

92

87

Per litter

11.5±3.1

11.2±3.9

12.1±3.1

11.9±1.9

11.0±3.3

Foetal weight (g)

Individual

4.7±0.3

4.8±0.4*

4.8±0.4*

4.7±0.4

4.1±0.5*

Litter

4.7±0.2

4.9±0.3

4.8±0.3

4.7±0.4

4.0±0.4*

Sex ratio (M/F)

139/130

70/82

115/102

160/140

85/80

a Proportions were analysed by the chi-square test. All other parameters were analysed by one-way analysis of variance and Student’s t-test. Values are mean ± SD.

*p < 0.05 v. controls.

Table 3: Signs of delayed ossification in foetuses of rats treated with alpha-terpinene on days 6-15 of pregnancy (a)

 

alpha-terpinene (mg/kg bw/day)

 

0

30

60

125

250

Foetuses examined

189

109

151

207

114

Foetuses with signs of delayed ossification (%)

11.1

14.7

53.0*

73.4*

88.6*

Foetuses (%) with retarded ossification in

Skull bones

0.5

4.6*

2.6

2.9*

16.6*

Vertebral column

1.6

0.9

22.5*

34.8*

21.0*

Sternum

11.6

5.5*

45.0*

70.0*

87.7*

Ribs

0

0

6.0*

13.5*

6.1*

Forelimbs

1.6

1.8

13.2*

9.2*

9.6*

Hindlimbs

4.8

9.2

37.7*

37.2*

47.4*

Signs of delayed ossification: not ossified (whole bone not stained); poorly ossified (whole bone is poorly ossified); and irregular spongy bones.

a Data were analysed by the chi-square test.

*p < 0.05 v. controls.

Table 4: Externally visible and visceral anomalies in foetuses of rats treated orally with alpha-terpinene on days 6-15 of pregnancy (a)

 

alpha-terpinene (mg/kg bw/day)

Treatment

0

30

60

125

250

External examination (no. of foetuses)

275

158

218

299

165

Foetuses with anomalies (%)

Haematoma

10 (3.6)

7 (4.4)

7 (3.2)

16 (5.3)

13 (7.9)

Tail

Bent end

1 (0.4)

0

2 (0.9)

6 (2.0)

4 (2.4)

Kinky

3 (1.1)

10 (6.3)*

3 (1.4)

6 (2.0)

5 (3.0)

Pale

0

0

1 (0.4)

0

0

Oedema

1 (0.4)

0

0

0

0

Irregular positioning of forepaws

0

1 (0.6)

0

4 (1.3)

0

Irregular positioning of hindpaws

2 (0.7)

2 (1.3)

1 (0.4)

3 (1.0)

2 (1.2)

Visceral examination (no. of foetuses)

86

49

67

92

51

Foetuses with anomalies (%)

Spleen (ectopic)

1 (1.2)

0

0

0

0

Heart (smaller)

0

1 (2.0)

0

0

0

Liver (smaller)

1 (1.2)

0

0

0

0

Adrenal gland (smaller)

0

0

1 (1.5)

0

0

Testes (ectopic)

3 (3.5)

0

1 (1.5)

1 (1.1)

0

Ureter (thicker)

0

0

0

0

1 (2.0)

a Proportions were analysed by the chi-square test or, alternatively, by Fischer’s exact test.

*p < 0.05 v. controls

Table 5: Foetal organ weight in rats treated orally with alpha-terpinene on days 6-15 of pregnancy (a)

 

alpha-terpinene (mg/kg bw/day)

Treatment

0

30

60

125

250

Foetuses examined

86

49

67

92

51

Foetal body weight (g)

4.9±0.5

5.3±0.5

5.3±0.4

5.2±0.5

4.2±0.5*

Foetal organ weights (mg)

Spleen

4.9±1.5

4.0±1.8

4.8±1.6

4.7±1.8

4.7±1.4

Heart

29.1±5.0

29.9±5.0

28.7±4.0

29.2±5.0

26.5±5.0*

Liver

370.0±66.0

372.0±48.0

375.0±39.0

362±80.0

335.0±64.0

Kidneys

Right

10.8±2.0

11.1±1.7

12.2±1.7*

12.1±2.4*

11.8±2.0*

Left

10.4±2.2

10.4±1.6

11.4±1.7*

11.1±2.0*

12.0±2.0*

Lung

143.0±18.0

139.0±12.0

142.0±14.0

138.0±15.0*

131.0±24.0*

Thymus

7.6±1.1

7.4±1.5

8.0±1.4

7.5±1.6

5.3±1.7*

a Data were analysed by one-way analysis of variance and Student’s t-test. Values are mean ± SD.

*p < 0.05 v. controls.

Table 6: Skeletal anomalies in foetuses of rats treated orally with alpha-terpinene on days 6-15 of pregnancy (a)

 

alpha-terpinene (mg/kg bw/day)

 

0

30

60

125

250

Foetuses examined

189

109

151

207

114

Foetuses with skeletal anomalies (%)

19.6

27.5

33.1

61.3

89.5

Foetuses (%) showing anomalies in:

Skull

5.3

8.2

16.5*

34.8*

63.1*

Os basisphenoid Bifurcated

0

0.9

0.7

0

0

Os basoccipitale Irregular shape

0

0

0

0

1.7

Os squamosum Irregular shape

4.8

6.4

13.2*

24.6*

35.1*

Os frontale Distance too large

0

0.9

0

0

0.9

Os interparietale Bone hole

0

0

0

0

0.9

Os palatinum Bone hole

0

0

1.3

1.4

0.9

Os parietale Distance too large

0

0.9

0

1.0

0.9

Os suproccipitale

Discontinuous

0

0

0

0

0.9

Gap

0

0

0

0.5

0.9

Incomplete ossification

0.5

0.9

2.6

12.6*

36.0*

Os tympanicum Discontinuous

0

0

0

0

0.9

Vertebral column

0

0.9

0

0

2.6

Atlas

Thicker

0

0

0

0

1.7

Cervical vertebra

Irregular shape

0

0

0

0

0.9

Fused

0

0

0

0

0.9

Thoracic vertebra

Fused with rib

0

0

0

0

0.9

Two ossification centra

0

0.9

0

0

0.9

Ribs

6.9

10.1

8.6

20.3*

53.5*

Shorter

5.8

6.4

6.0

19.8*

50.0*

Extra

Cervical

0.5

0.9

1.3

1.0

7.0*

Lumbar

0.5

2.7

1.3

1.0

0.9

Sternum

5.8

5.5

3.3

8.2

11.4*

Dislocated

5.8

5.5

3.3

8.2

11.4*

Forelimbs

2.6

5.5

6.6

17.9*

6.1

Irregular position

0.5

0.9

0

2.9

0

Os processus deltoid

Bone hole

1.6

0

1.3

1.9

3.5

Irregular shape

0.5

4.6

6.6

14.5

2.6

a Data were analysed by the chi-square test.

*p < 0.05 v. controls.

Conclusions:
Under the test conditions, alpha terpinene can adversely affect embryofoetal development in the rat at oral doses higher than 60 mg/kg body weight and is toxic to the mother at oral doses higher than 125 mg/kg body weight. Thus, the NOAEL of alpha terpinene for embryofoetal toxicity was determined to be 30 mg/kg body weight.
Executive summary:

A pre-natal developmental toxicity test was performed with alpha terpinene following a method equivalent to OECD Guideline 414. Alpha terpinene dissolved in corn oil at doses of 30, 60, 125 and 250 mg/kg body weight was given by gavage to female Wistar rats from day 6 to 15 of pregnancy. Caesarean sections were performed on day 21 of pregnancy. The number of implantation sites, living and dead foetuses, resorptions and corpora lutea were recorded. All foetuses were weighed and examined for externally visible malformations. One-third of the foetuses of each litter were evaluated for visceral anomalies. The remaining foetuses were examined for skeletal malformations. A reduction in body weight minus uterine weight at term indicated that the two highest doses tested (125 and 250mg/kg bw) were maternally toxic. No increase in the ratio of resorptions/implantations was observed over the dose range tested. The highest dose (250 mg/kg bw) reduced the ratio of pregnant/treated female. A decrease in foetal body weight and an increase in foetal kidney weights were noted at 250 mg /kg bw. Signs of delayed ossification (poorly ossified and not ossified bones as well as irregular spongy bones) and a higher incidence of minor skeletal malformations were observed at doses of 60 mg/kg bw or more. These findings indicate that the NOAEL for alpha terpinene-induced embryofoetotoxicity can be set at 30 mg/kg bw by the oral route.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(The study does not provide specified information on abnormalities findings)
GLP compliance:
not specified
Remarks:
No information reported
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Department of Physiology and Pharmacology of Federal University of Pernambuco (UFPE,
Pernambuco, Brazil)
- Females (if applicable) nulliparous and non-pregnant: yes
- Diet (e.g. ad libitum): industrialized dry food (Presence®, Purina, Brazil), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 ºC
- Humidity (%): 55-65 %
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
other: Tween-80 aqueous solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
1,8-cineole (CIN) was emulsified in a 1% Tween-80 aqueous solution before administration to the
animals.
Volume administered: 10 mL/kg bw

VEHICLE
Tween-80 (CAS No: 9005-65-6) was obtained from Sigma-Aldrich® (St. Louis, MO, USA).
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 2 female/1 male
- Length of cohabitation: nulliparous female rats were distributed in polyethylene cages, into contact with a male rat during the dark phase of the environmental cycle. The next day, male rats were removed from the cages and the female vaginal lavage was carried out with 0.9% physiological saline solution.
- Proof of pregnancy: The spermatozoids viewing associated with the diagnosis of the estrous phase of the estral cycle was considered indicative of pregnancy (Cooper et al., 1993). The observation of the presence of sperm in the vaginal smear has defined the 1st day of pregnancy.
Duration of treatment / exposure:
Preimplantation period: From 1st to 6th day of pregnancy.
Organogenic period: From 7th to 14th day of pregnancy.
Frequency of treatment:
Daily
Duration of test:
From 1st to 21st day of pregnancy
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control (Group I)
Dose / conc.:
250 mg/kg bw/day
Remarks:
Group II
Dose / conc.:
500 mg/kg bw/day
Remarks:
Group III
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Group IV
No. of animals per sex per dose:
Preimplantation period: 9-10
Organogenic period: 7-9
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- During pregnancy, the rats were evaluated for survival, altered appearance and any clinical signs of toxicity, such as changes in food and water intake, piloerection, diarrhea, changes in locomotor activity and vaginal bleeding.

BODY WEIGHT: Yes
- Time schedule for examinations: daily.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: uterine horns, ovaries, fetuses and placentae

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: The fetuses and placentae were weighed (absolute mass).
- Number of corpora lutea: Yes.
- Number of implantations: Yes.
- Number of resorptions: Yes.
- Other:
Ovaries (right and left) were weighed
Fetal examinations:
Live and dead fetuses were recorded.
The fetuses were observed macroscopically for any visible abnormalities.
Statistics:
Values were expressed as mean ± standard error of mean (S.E.M.) and the differences are analyzed by variance analysis (ANOVA) followed by Dunnett’s test or Student’s T test for unpaired samples. The implantation and resorption indexes, as well as pre-implantation and post-implantation loss rates, were analyzed using the Kruskal–Wallis test followed by the Dunn test, when necessary.The level of significance for rejection of the null hypothesis was set at 5% (p < 0.05). Statistical analyses were performed using GraphPad Prism 5.0®.
Indices:
Implantation index: total number of implantation sites/total number of corpora lutea × 100)
Resorption index: total number of resorption sites/total number of implantation sites × 100
Preimplantation loss rate: (number of corpora lutea − number of viable implantations)/number of corpora lutea × 100
Post-implantation loss rate: (number of implantations − number of live fetuses)/number of implantations × 100
Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs of toxicity, such as salivation, piloerection, diarrhea, changes in locomotor activity or changes in behavior at any of the doses administered.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
Treatment at any dose produced no deaths in pregnant rats treated during pre-implantation or organogenesis.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant decrease was observed in maternal weight gain during pre-implantation (1st to 6th day) and organogenesis (7th to 14th day) in females treated with all doses, and during pregnancy (1st to 20th day) in females treated with 1000 mg/kg during the preimplantation period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No changes in the intake of food were observed during pregnancy in either period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No changes in the intake of water were observed during pregnancy in either period.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
During the pre-implantation or organogenesis periods, the ovary and placental masses were similar in all treated experimental groups.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
During the pre-implantation or organogenesis periods, the implantation index and the loss rate of pre-and post-implantation were similar in all treated experimental groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
During the pre-implantation or organogenesis periods, the resorption index was similar in all treated experimental groups.
Early or late resorptions:
no effects observed
Description (incidence and severity):
During the pre-implantation or organogenesis periods, the resorption index was similar in all treated experimental groups.
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
Dead fetuses were observed during the pre-implantation and organogenesis periods at doses of 500 and 1000 mg/kg, and also in the control group. Since there was no reduction in the mass of fetuses in animals treated with 1000 mg/kg during the organogenesis period, it can be inferred that there is a relation between reduced maternal body mass gain and a reduction in the mass of rat foetuses treated with this dose during the pre-implantation period, but that needs to be better investigate.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
During the organogenesis period, a single rat at a dose of 1000 mg/kg presented vaginal bleeding on the 13th day of gestation and laparotomy did not reveal either live or dead fetuses.
There was a reduction in the number of corpora lutea in females treated with 250 mg/kg during the organogenesis, when compared to the control group.
Key result
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduction in the mass of fetuses were observed in females treated with 1000 mg/kg in the preimplantation period.
Since there was no reduction in the mass of fetuses in animals treated with 1000 mg/kg during the organogenesis period, it can be inferred that there is a relation between reduced maternal body mass gain and a reduction in the mass of rat foetuses treated
with this dose during the pre-implantation period, but that needs to be better investigate.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Remarks on result:
other: Due to the reduction of maternal body mass induced at the dose of 250 mg/kg in the pre-implantation and organogenesis periods, it was not possible to determine the NOAEL.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1: Reproductive parameters of female Wistar rats treated with 1,8-cineole (CIN) from 1st to the 6th day of pregnancy (preimplantation period).

Reproductive parameters

control

250 mg/kg

500 mg/kg

1000 mg/kg

Pregnant rats    

9

10

10

10

Mass gain in the pre-implantation period (g)a

15.29±1.51

-1.98±2.93*

0.66±3.53*

-6.12±3.73*

Mass gain in the pregnancy period (g)a

73.82±7.53

73.04±6.53

71.14±3.64

50.12±7.57*

Number of live fetuses  

96

116

117

84

Number of dead fetuses  

1

0

0

4

Offspring/dam relationshipa    

10.67±0.88

11.60±1.05

11.60±0.52

8.80±1.45

Fetuses mass (g)a   

2.35±0.06

2.32±0.10

2.29±0.071.60

1.60±0.28*

Placentae mass (g)a   

0.51±0.02

0.47±0.01

0.45±0.01

0.42±0.05

Ovary mass (mg/100g)a   

22.87±0.87

18.70±1.25

22.16±1.83

20.96±1.63

Number of implantation sites  

107

123

121

94

Number of resorption sites  

10

7

4

6

Number of corpora luteaa  

12.89±0.54

12.70±1.11

12.10±0.43

11.80±0.99

Implantation index (%)b   

100

100

100

89.96

Resorption index (%)b   

0

0

0

6.67

Pre-implantation loss (%)b   

0

0

0

10.05

Post-implantation loss (%)b   

0

0

0

3.33

Implantation index (total number of implantation sites/total number of corpora lutea × 100), resorption index (total number of resorption sites/total number of implantation sites × 100, pre-implantation loss rate (number of corpora lutea − number of viable implantations/number of corpora lutea × 100) and post-implantation loss rate (number of implantations − number of live fetuses/number of implantations × 100). The values are expressed as mean ± S.E.M.(a) or median (b). *Statistically different from control group (ANOVA followed by Dunnett's test, p < 0.05).

Table 2: Reproductive parameters of female Wistar rats treated with 1,8-cineole (CIN) from 7th to the 14th day of pregnancy (organogenic period).

Reproductive parameters

control

250 mg/kg

500 mg/kg

1000 mg/kg

Pregnant rats    

8

9

8

7

Mass gain in the organogenic period (g)a

20.05±1.60

-3.00±3.22*

-1.60±4.20*

0.37±8.89*

Mass gain in the pregnancy period (g)a

74.79±5.13

56.67±4.72

54.91±9.38

57.17±10.34

Number of live fetuses  

85

90

81

62

Number of dead fetuses  

1

0

1

0

Offspring/dam relationshipa    

10.63±0.84

10.00±0.60

10.13±1.10

8.85±1.33

Fetuses mass (g)a   

2.33±0.01

2.42±0.12

2.34±0.18

2.34±0.23

Placentae mass (g)a   

0.47±0.030.47

±0.010.49

±0.020.50

0.50±0.03

Ovary mass (mg/100g)a   

24.69±1.87

23.45±1.47

24.77±2.36

25.99±2.38

Number of implantation sites  

91

90

80

64

Number of resorption sites  

7

10

3

12

Number of corpora luteaa  

13.75±0.83

11.11±0.48*

11.88±0.74

12.14±0.96

Implantation index (%)b   

78.3

100

88.31

78.57

Resorption index (%)b   

6.25

8.33

0

0

Pre-implantation loss (%)b   

21.27

0

11.69

21.43

Post-implantation loss (%)b   

0

8.33

0

0

Implantation index (total number of implantation sites/total number of corpora lutea × 100), resorption index (total number of resorption sites/total number of implantation sites × 100, pre-implantation loss rate (number of corpora lutea − number of viable implantations/number of corpora lutea × 100) and post-implantation loss rate (number of implantations − number of live fetuses/number of implantations × 100). The values are expressed as mean ± S.E.M.(a) or median (b). *Statistically different from control group (ANOVA followed by Dunnett's test, p < 0.05).

Conclusions:
In a reproductive toxicity study performed by oral route (gavage) with 1,8-cineole using Wistar rats, the results obtained provide evidence that possibly the test substance presents maternal and fetal toxicity, but it is concluded that further studies need to be conducted. The NOAEL was not established since effects in maternal body weight were found at the lowest dose tested (250 mg/kg bw/day).
Executive summary:

A reproductive toxicity study was performed by oral route (gavage) with 1,8-cineole using female Wistar rats. The pregnant rats were randomly distributed into 8 groups (n = 7-10 per group). 4 groups were assigned to treatment during the pre-implantation period (1st to 6th day of pregnancy) and 4 groups to treatment during organogenesis (7th to 14th day of pregnancy). Test substance was administered to all animals of both pregnancy periods with doses of 0 (control, consisted only in 1% Tween-80 aqueous solution), 250, 500 and 1000 mg / kg bw/day.

The test substance produced no deaths or signs of toxicity in pregnant rats treated during the pre-implantation or organogenesis periods. No alterations were observed in maternal food and water intake during the course of pregnancy in both periods, although, during the pre-implantation period, a reduction in body mass gain was observed in females at all doses. After discontinuation of this treatment, the situation was reversed, as evidenced by the lack of difference in body mass gain during pregnancy (1st to 20th day). A similar response was observed during organogenesis. With regard to reproductive parameters, the data showed no significant differences in the number of fetuses, placentae and ovary masses, implantation or resorption index, or pre-implantation and post-implantation losses.

Dead fetuses were observed during the pre-implantation and organogenesis periods at doses of 500 and 1000 mg/kg, and also in the control group. Since there was no reduction in the mass of fetuses in animals treated with 1000 mg/kg during the organogenesis period, it can be inferred that there is a relation between reduced maternal body mass gain and a reduction in the mass of rat foetuses treated with this dose during the pre-implantation period, but that needs to be better investigate.

These results provide evidence that possibly the test substance presents maternal and fetal toxicity, but it is concluded that further studies need to be conducted. The NOAEL was not established since effects in maternal body weight were found at the lowest dose tested (250 mg/kg bw/day).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
591 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A weight of evidence approach has been applied. Several experimental studies are available with a Klimisch score of 2.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Weight of evidence from experimental results with individual main components:

A pre-natal developmental toxicity test was performed with camphene according to OECD Guideline 414. 20 pregnant Sprague-Dawley rats were orally exposed by gavage during the 'critical' phase of organogenesis (daily from the 6th to 15th day of pregnancy). Tested concentrations were 0, 250 and 1000 mg/kg bw/day, using sesame oil as vehicle. No substance-related mortality was observed in the dams. Six of the 20 dams treated with 1000 mg/ kg bw/day showed reduced motor activity and salivation. No other clinical signs were observed in the remaining high-dosed and the low-dosed dams. Body weights remained within the normal range and body weight gain showed no influence of the test compound. Transient impairment of the food consumption by the highest tested dose was observed. Treatment did not influence drinking-water consumption. No substance-related pathological changes were detected at autopsy. The highest tested dose caused a slight but not significant increase of the resorption rate and, consequently, of the post-implantation loss. No further influence on the prenatal development was detected. All other fetal parameters were within the normal range of the control group. External macroscopic inspection, examination of soft tissue and skeletal examination revealed no substance-related variations and/or retardations. No dead fetuses occurred in the substance treated and control dams. Under these test conditions, the NOEL for the dams and for the fetal organism was 250 mg camphene/kg bw/day. Camphene did not possess teratogenic properties.

In a prenatal developmental toxicity study, d-limonene was administered orally to groups of pregnant Japanese white rabbits at dose levels of 250, 500 and 1000 mg/kg bw/day for 13 days from Day 6 to 18 of gestation.External examination of fetuses showed no anormalies. Visceral and skeletal examinations revealed some anormalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae but these effects were not dose-dependent. D-limonene was not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity was considered to be greater than 1000 mg/kg bw/day.

In another prenatal developmental toxicity study, d-limonene was administered orally to groups of pregnant ICR mice (20/dose: 15 for teratogenicity study, 5 for postnatal development) at dose levels of 0, 591 and 2363 mg/kg bw/day for 6 days from Day 7 to 12 of gestation.A significant decrease of bodyweight gain in pregnant mice was observed at 2363 mg/kg bw/day. An incidence of lumber rib and fused rib in the fetuses increased significantly at 2363 mg/kg bw/day compared with those of control. In the observation of skeletal development in fetuses, retarded ossification of proximal phalanx of fore limb, metatarsal bone and proximal phalanx of hind limb were observed but were not dose-dependent or only observed at the highest dose, associated with maternal toxicity. According to these results, the NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day.

In a third developmental toxicity study, d-limonene was administered orally to groups of pregnant Wistar rats (20/dose: 15 for teratogenicity study, 5 for postnatal development) at dose levels of 0, 591 and 2869 mg/kg bw/day for 7 days from Day 9 to 15 of gestation.At 2869 mg/kg bw/day, maternal bodyweight decreased and several mothers (40%) died during treatment. Delayed ossification of fetuses metacarpal bone and proximal phalanx at 2869 mg/kg bw/day was increased and adecreased tendency of bodyweight was noted in postnatal male offsprings born to mothers treated at 2869 mg/kg bw/day, compared with the control group. Thymus, spleen and ovaries weights decreased in offsprings born to mothers treated at 2869 mg/kg. According to these results, the NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day.

A pre-natal developmental toxicity test was performed with alpha terpinene following a method equivalent to OECD Guideline 414. Alpha terpinene dissolved in corn oil at doses of 30, 60, 125 and 250 mg/kg body weight was given by gavage to female Wistar rats from day 6 to 15 of pregnancy. Caesarean sections were performed on day 21 of pregnancy. The number of implantation sites, living and dead foetuses, resorptions and corpora lutea were recorded. All foetuses were weighed and examined for externally visible malformations. One-third of the foetuses of each litter were evaluated for visceral anomalies. The remaining foetuses were examined for skeletal malformations. A reduction in body weight minus uterine weight at term indicated that the two highest doses tested (125 and 250mg/kg bw) were maternally toxic. No increase in the ratio of resorptions/implantations was observed over the dose range tested. The highest dose (250 mg/kg bw) reduced the ratio of pregnant/treated female. A decrease in foetal body weight and an increase in foetal kidney weights were noted at 250 mg /kg bw. Signs of delayed ossification (poorly ossified and not ossified bones as well as irregular spongy bones) and a higher incidence of minor skeletal malformations were observed at doses of 60 mg/kg bw or more. These findings indicate that the NOAEL for alpha terpinene-induced embryofoetotoxicity can be set at 30 mg/kg bw by the oral route.

A reproductive toxicity study was performed by oral route (gavage) with 1,8-cineole using female Wistar rats. The pregnant rats were randomly distributed into 8 groups (n = 7-10 per group). 4 groups were assigned to treatment during the pre-implantation period (1st to 6th day of pregnancy) and 4 groups to treatment during organogenesis (7th to 14th day of pregnancy). Test substance was administered to all animals of both pregnancy periods with doses of 0 (control, consisted only in 1% Tween-80 aqueous solution), 250, 500 and 1000 mg / kg bw/day.

The test substance produced no deaths or signs of toxicity in pregnant rats treated during the pre-implantation or organogenesis periods. No alterations were observed in maternal food and water intake during the course of pregnancy in both periods, although, during the pre-implantation period, a reduction in body mass gain was observed in females at all doses. After discontinuation of this treatment, the situation was reversed, as evidenced by the lack of difference in body mass gain during pregnancy (1st to 20th day). A similar response was observed during organogenesis. With regard to reproductive parameters, the data showed no significant differences in the number of fetuses, placentae and ovary masses, implantation or resorption index, or pre-implantation and post-implantation losses. Dead fetuses were observed during the pre-implantation and organogenesis periods at doses of 500 and 1000 mg/kg, and also in the control group. Since there was no reduction in the mass of fetuses in animals treated with 1000 mg/kg during the organogenesis period, it can be inferred that there is a relation between reduced maternal body mass gain and a reduction in the mass of rat foetuses treated with this dose during the pre-implantation period, but that needs to be better investigate. These results provide evidence that possibly the test substance presents maternal and fetal toxicity, but it is concluded that further studies need to be conducted. The NOAEL was not established since effects in maternal body weight were found at the lowest dose tested (250 mg/kg bw/day).

Conclusion for the test substance:

Experimental results from studies performed with the main components camphene and d-limonene have been selected in order to decide on the overall assessment of the test substance. Camphene was not teratogenic in rat fetuses and the NOAEL for fetal toxicity was considered to be greater than the highest dose tested. Also, d-limonene was not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity was considered to be greater than the highest dose tested. In two developmental toxicity studies with d-limonene in rats and mouse, slight ossification delays/malformations and organ weights changes were observed but not dose-related and/or observed at doses where maternal toxicity was identified. According to these results, the NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day.

Justification for classification or non-classification

Based on the available information, the substance is not classified for toxicity to reproduction in accordance with CLP Regulation (EC) no 1272/2008.

Justification for not classifying the substance based on the component alpha terpinene results from a pre-natal developmental toxicity test

Alpha-terpinene (30, 60, 125 and 250 mg/kg body weight) in corn oil was given by gavage to female Wistar rats from day 6 to 15 of pregnancy. Caesarean sections were performed on day 21 of pregnancy. A reduction in body weight minus uterine weight at term indicated that the two highest doses tested (125 and 250mg/kg body weight orally) were maternally toxic. The highest dose of 250 mg/kg body weight reduced the ratio of pregnant/treated female.

It should be taken into account that marked reductions in overall pregnancy weight gain minus uterine weight at term clearly indicate that the two highest doses of 125 and 250 mg/kg body weight are maternally toxic. The significant reduction of the ratio of pregnant/ sperm-positive females was only observed at the highest dose tested, i.e. it is unclear whether the decrease in pregnant females is secondary to the maternal toxicity. As there are no repeated dose toxicity studies which could indicate more specific effects, it cannot be excluded that alpha-terpinene also induces other maternal effects that were not determined in this developmental study. Therefore, it cannot be excluded that the observed maternal reproductive effects are secondary to general maternal toxicity.

The CLP Regulation (EC) no. 1272/2008 clearly states that the effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects.

On the other hand, regarding the fetal effects observed, for the 250 mg/kg bw/day group, the reduction in mean foetal weight would also contribute to an alteration in the rate of ossification. However, there is no reduction in mean foetal weight at 125 mg/kg bw/day. The publication lacks key information which could be used to better understand the reasons for the apparent increase in occurrence of these altered areas of ossification. It is possible that a few litters of lower weight foetuses are contributing to the elevated incidences, but unfortunately no litter based information is reported. In addition, there is no information provided for the range of normal variation in frequency of occurrence in control foetuses (i.e. historical control data). Despite the omission of these aids to understanding, it can be argued that there is an effect of treatment on foetal ossification at 125 and 250 mg/kg bw/day but given the number of ossification centres affected this effect is minimal and also, is transient in nature. In the absence of an effect of 125 mg/kg bw/day on foetal weight it could be reasonably argued that the changes in ossification are too minimal to be considered indicative of developmental toxicity per se, i.e., they are of no toxicological significance. However, additional data is needed to confirm the (absence of) observed effects.

For the 60 mg/kg bw/day group, only one area of the foetal skeleton is seen to be less well ossified in comparison with the controls. Although apparently dose-related, in isolation this single finding should not be considered to represent developmental toxicity due to 60 mg/kg bw/day.

The effect on embryofoetal development is incorrectly described as adverse, the changes seen do not have an effect on the foetus, they only represent an alteration in the timing of ossification, a transient process in itself, and affect only very few of the many ossification centres. Furthermore, 60 mg/kg bw/day should not be considered as an effect level for developmental toxicity on the basis of the appearance of one ossification centre only.

In conclusion, there is no evidence that alpha-terpinene has teratogenic or embryotoxic effects in the absence of maternal toxicity and thus, the classification for Toxicity to Reproduction should not be warranted.