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Key value for chemical safety assessment

Effects on fertility

Description of key information

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

Link to relevant study records
Reference
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 REACH Annex VIII, column 2, the study does not need to be conducted because pre-natal developmental toxicity studies are available.


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

Effects on developmental toxicity

Description of key information

Developmental toxicity: Weight of evidence and read-across approach:

In a developmental toxicity study (Araujo, 1996) alpha terpinene was given by gavage to female Wistar rats from day 6 to 15 of pregnancy in doses of 30, 60, 125 and 250 mg/kg body weight). A NOAEL of alpha terpinene for embryofoetal toxicity was determined to be 30 mg/kg bw.

At the highest dose of 250 mg/kg bw there was a reduction in the ratio of pregnant/ sperm-positive females. However, a reduction in body weight minus uterine weight at term clearly indicates that the two highest doses tested (125 and 250 mg/kg bw) were 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. In this respect, 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.

In addition, the lowest proportion of sperm positive, but not pregnant females, amounting to 4 %, was observed at the dose of 125 mg/kg bw, indicating that a reduction in proportion of sperm positive females without any implantation was not dose-related. The reduction of sperm positive females without any implantation only at the top dose, without dose-response relationship, cannot be taken reliably as treatment-related since the procedure to assign sperm positive females to treatment groups was not reported to be randomised, and the fertility of males used for insemination was not ascertained.

Furthermore, regarding the fetal effects observed, for a few areas of the foetal skeleton there was some indication of a dose-related effect on ossification. 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. Thus, it can be assumed 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.

Therefore, 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, from this study 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. This conclusion is supported by the opinion of the Committee for Risk Assessment (ECHA) on a dossier proposing harmonised classification and labelling for alpha terpinene (CLH-O-0000001412-86-274/F, adopted on 15 march 2019).

Similar effects have been observed with the analogue substance d-limonene.

In rats, the oral administration of d-limonene (2869 mg/kg body weight per day) on days 9-15 of gestation resulted in decreased body weight and deaths among the dams. Delayed ossification and decreased total body and organ weights (thymus, spleen, and ovary) were observed in the offspring (Tsuji, 1975). In mice, the oral administration of d-limonene (2363 mg/kg body weight per day) on days 7-12 of gestation resulted in reduced growth in the mothers and a significantly increased incidence of skeletal anomalies and delayed ossification in the offspring (Kodama, 1977a). The oral administration of d-limonene (250, 500, or 1000 mg/kg body weight per day) to rabbits on days 6–18 of gestation had no dose-related effects on the offspring. At the highest dose, there were some deaths and reduced weight gain among the dams; at the intermediate dose, growth was decreased (Kodama, 1977b).

The CICAD on Limonene (Concise International Chemical Assessment Document 5 (CICAD). Limonene. WHO, Geneva 1998) stated that there is no evidence that limonene has teratogenic or embryotoxic effects in the absence of maternal toxicity, concluding that the substance is essentially non-toxic for human health hazards.

Moreover, the European Food Safety Authority made a review of the toxicological properties of d-limonene in 2010, and concluded on the absence of safety concern due to d-limonene intake, with no specific concern related to the absence of any study for toxicity to reproduction for this substance (EFSA, 2010. Scientific Opinion on Flavouring Group Evaluation 25Rev1: Aliphatic and aromatic hydrocarbons from chemical group 31. EFSA Journal 2010; 8(5): 1334).

Conclusion for the test substance:

Experimental results from studies performed with alpha terpinene and the analogue substance d-limonene have been evaluated in order to decide on the overall assessment of the test substance alpha terpinene. After evaluation of the available study, alpha terpinene is considered not teratogenic in rat fetuses and the NOAEL for fetal toxicity is considered to be the highest dose tested (250 mg/kg bw/day). In addition, alpha terpinene was found maternally toxic based on the reduction in body weight minus uterine weight at 125 and 250 mg/kg bw. However, a NOAEL of 250 mg/kg bw/day for maternal toxicity could be considered taking into account that no other signs of maternal toxicity were noted and no gross pathological alteration in the maternal organs of any group was found at caesarian section. 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 other 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 of d-limonene for maternal and fetal toxicity was considered to be 591 mg/kg bw/day.

Based on all the above information, weight of evidence and read across approach was applied, and it is concluded that alpha terpinene should not be classified for Toxicity to Reproduction and a NOAEL for developmental toxicity could be considered to be 250 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:
guideline study with acceptable restrictions
Remarks:
Test method equivalent to OECD guideline 414
Qualifier:
equivalent or similar to guideline
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 or test system 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:
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 or test system 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 or test system 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
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 or test system 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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
591 mg/kg bw/day (actual dose received)
Based on:
other: Read across from an analogue
Basis for effect level:
other: maternal toxicity
Remarks on result:
other: read-across from an analogue for which NOAEL = 591 mg/kg bw/day.
Key result
Abnormalities:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
591 mg/kg bw/day
Based on:
other: Read across from an analogue
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Remarks on result:
other: read-across from an analogue for which NOAEL = 591 mg/kg bw/day.
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: rib
Description (incidence and severity):
Read across from an analogue for which Increase in incidence of lumber rib and fused rib compared to control were observed.
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
Conclusions:
Based on the read-across approach from experimental data on the analogue d-limonene, the NOAEL of alpha terpinene for maternal and fetal toxicity can be 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.

Based on these results, the read-across approach was applied and the NOAEL of alpha terpinene for maternal and fetal toxicity can be 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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
other: Read across from an analogue
Basis for effect level:
other: maternal toxicity
Remarks on result:
other: read-across from an analogue for which NOAEL = 250 mg/kg bw/day
Key result
Abnormalities:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
other: Read across from an analogue
Basis for effect level:
other: fetotoxicity
Remarks on result:
other: read-across from an analogue for which NOAEL > 1000 mg/kg bw/day.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Based on the read-across approach from experimental data on the analogue d-limonene, alpha terpinene can be regarded as not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity is considered to be greater than 1000 mg/kg bw/day. The NOAEL for maternal toxicity is 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. Based on these results, the read-across approach was applied and alpha terpinene can be regarded as not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity is considered to be greater than 1000 mg/kg bw/day. The NOAEL for maternal toxicity is considered to be 250 mg/kg bw/day based on the decreased bodyweight gain.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
591 mg/kg bw/day (actual dose received)
Based on:
other: Read across from an analogue
Basis for effect level:
other: maternal toxicity
Remarks on result:
other: read-across from an analogue for which NOAEL = 591 mg/kg bw/day.
Key result
Abnormalities:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
591 mg/kg bw/day
Based on:
other: Read across from an analogue
Sex:
male/female
Basis for effect level:
skeletal malformations
other: read across from an analogue for which decreased bodyweight gain (male offsprings) and organ weights at 2869 mg/kg bw/day were observed.
Remarks on result:
other: read-across from an analogue for which NOAEL = 591 mg/kg bw/day.
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: skeletal: metacarpals and proximal phalanges
Description (incidence and severity):
read across from an analogue for which prolongation of the ossification of metacarpals and proximal phalanges were observed.
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
Conclusions:
Based on the read-across approach from experimental data on the analogue d-limonene, the NOAEL of alpha terpinene for maternal toxicity can be considered to be 591 mg/kg bw/day based on the deaths and decreased bodyweight gain. The NOAEL for fetal toxicity is 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.

 

Based on these results, the read-across approach was applied and the NOAEL of alpha terpinene for maternal toxicity can be considered to be 591 mg/kg bw/day based on the deaths and decreased bodyweight gain. The NOAEL for fetal toxicity is considered to be 591 mg/kg bw/day based on the delayed skeletal formation and decreased bodyweight gain.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A weight of evidence and read across approach has been applied. Several experimental studies with alpha terpinene and analogue substance d-limonene are available with a Klimisch score of 2 in rats, mice and rabbits.
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

Summary of results of the experimental studies used to assess the reproductive toxicity of alpha terpinene:

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 (Araujo, 1996). 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.

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 for 7 days from Day 9 to 15 of gestation (Tsuji, 1975). 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 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. According to these results, the NOAEL for maternal and fetal toxicity was considered to be 591 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 (Kodama, 1977a). 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 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 (Kodama, 1977b). 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.

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.

Additional information