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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1996

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
p-mentha-1,3-diene
EC Number:
202-795-1
EC Name:
p-mentha-1,3-diene
Cas Number:
99-86-5
Molecular formula:
C10H16
IUPAC Name:
1-isopropyl-4-methylcyclohexa-1,3-diene

Test animals

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).


Administration / exposure

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)
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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

Effect levels (maternal animals)

open allclose all
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

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

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

Fetal abnormalities

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.

Overall developmental toxicity

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.