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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The reliability is based on read across for a structural analogue compound, myrcene (CAS 123-35-3). See read across justification section 13 The myrcene study report was conclusive and done following protocol similar to OECD 414 with minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Justification for type of information:
Myrcene and Farnesene have similar chemical structures and therefore are expected to have similar physical/chemical characteristics. Thus, read across of myrcene data is a reasonable approach.
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The reliability is based on read across for a structural analogue compound, myrcene (CAS 123-35-3). See read across justification section 13 The myrcene study report was conclusive and done following protocol similar to OECD 414 with minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Justification for type of information:
Myrcene and Farnesene have similar chemical structures and therefore are expected to have similar physical/chemical characteristics. Thus, read across of myrcene data is a reasonable approach.
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
mating conditions (1 male for 3 females); food consumption not followed; bodyweight only recorded at Day 0, 6, 16 and 20 of gestation
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Oswaldo Cruz Foundation Central Animal House breeding stock
- Housing: Housed in plastic cages with wood shavings as bedding
- Diet (e.g. ad libitum): Nuvital diet for laboratory animals, Nuvilab Ltd, Curitiba, Brazil; ad libitum
- Water (e.g. ad libitum): Tap water; ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23 °C
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test material was dissolved in corn oil
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
None
Details on mating procedure:
Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:3
- Length of cohabitation: 2 h
- Proof of mating: Sperm in vaginal smear referred to as Day 0 of pregnancy
Duration of treatment / exposure:
10 days (Day 6-15 of pregnancy)
Frequency of treatment:
once daily
Duration of test:
20 days
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 200 mg/kg bw/day
No. of animals per sex per dose:
Treated females: 22-36; pregnant females: 16-29
Control animals:
yes, concurrent no treatment
Details on study design:
Control animals: as no statistically significant difference was found between the untreated and the vehicle treated control animals, in any of the parameters analysed, the data of both groups were pooled and were thereafter designated the control group.
Maternal examinations:
BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 16 and 20 of pregnancy
Ovaries and uterine content:
The uterine content was examined after termination: Yes, animals were sacrificed on Day 20 for caesarean examinations
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of living and dead foetuses: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:

- External examinations: Yes [all living foetuses]; weighed and examined for externally visible malformations
- Soft tissue examinations: Yes [5-7 litters per group]: evaluated for visceral malformations; heart, lungs, thymus, liver, spleen and kidneys were weighed and microdissected
- Skeletal examinations: Yes [all the remaining litters]
Statistics:

- Statistical analysis was by one way analysis of variance or, alternatively, by the Kruskal-Wallis test whenever the data did not fit a normal distribution
- Statistically significant differences between groups were tested by using a two sided Student's t-test or the Mann-Whitney U-test
- Proportions were analysed by the chi-square test
- Difference was considered to be statistically significant at P < 0.05
Indices:
na
Historical control data:
na
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: significant decreased body weight gain (1200 mg/kg)

Details on maternal toxic effects:
At 250 and 500 mg/kg bw /day: No treatment-related effects

At 1200 mg/kg bw/day:
- Mortality: 1/29 dams died on Day 11 of pregnancy
- Body weights: Statistically significant decrease in weight gain during days 6-11 of pregnancy (17.3 ± 7 g vs. 3.2 ± 13.2 g in control vs. 1200 mg/kg group, respectively)
- Cesarean examination: Number of visible implantation sites was significantly reduced (12.6 ± 2.2 vs. 10.2 ± 2.9 in control vs. 1200 mg/kg group, respectively)
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:

- Body weight: Treatment-related effects on foetal weight were only very slight, or even insignificant, if the litter is taken as the statistical unit of analysis
- External examinations: Increased frequency of irregularly positioned hind paws was observed in 1200 mg/kg bw/day group
- Visceral examinations: Visceral malformations such as enlarged ureters associated with an enlarged renal pelvis (one control foetus and one in 1200 mg/kg group), shorter ureter (one control foetus and one in 250 mg/kg bw/day group) and accessory (seventh) lobe in the liver (three foetus in 250 mg/kg bw/day group)
- Skeletal examinations: Increased incidence of delayed ossification and minor gross structural anomalies in the foetal skeleton were observed in 1200 mg/kg bw/day group
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental toxicity
Abnormalities:
effects observed, treatment-related
Localisation:
other: signs of retardation and of anomalies in the foetal skeleton observed at 1200 mg/kg bw/day
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 200 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Table 1. Occurrence of delayed ossification in foetuses of rats treated orally with β-myrcene on Days 6-15 of pregnancy

 

β-Myrcene (mg/kg bw)

Treatment

0

250

500

1200

Foetuses examined (no.)

114

106

116

209

Percentage of foetuses with signs of delayed ossificationαin:

Skull bones

4.4

0.9

3.4

9.6*

Caudal vertebrae

7

2.8

6

37.8*

Forelimbs/metacarpus

2.6

0

0.9

9.1*

Hind limbs/metatarsus

5.3

2.8

3.4

29.2*

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

* P < 0.05; chi-square test

Conclusions:
In a read-across study, the no-observed-adverse-effect level (NOAEL) for maternal toxicity and for embryo-foetotoxicity of β-myrcene administered orally (gavage) was reported to be 500 mg/kg bw/day in Wistar rats.
Executive summary:

A study was conducted to evaluate the embryo-foetotoxic potential of β-myrcene in the Wistar rats similarly to OECD Guideline 414.

β -Myrcene (250, 500 and 1200 mg/kg bw/day) in corn oil was given orally to Wistar rats from Day 6 to 15 of pregnancy. Two control groups, one received vehicle only and another without treatment, were also studied simultaneously. All rats were weighed on Day 0, 6, 16 and 20 of pregnancy. Caesarean sections were performed on Day 20 of pregnancy, and the number of resorptions and implantation sites were recorded. Foetuses were weighed and examined for external, visceral and skeletal malformations.

A significant effect (decrease) on maternal body weight gain (~ 82%%) was seen during gestation days 6 to 11, at 1200 mg/kg. There was also an increased incidence of delayed foetal ossification in the 1200 mg/kg dose group. At lower dose levels no maternal or foetal effects were observed that were considered to be related to treatment. The possibility that the delay in skeletal ossification seen in foetuses at 1200 mg/kg may have been caused indirectly by the significant maternal toxicity cannot be precluded.

In conclusion, the no-observed-adverse-effect level (NOAEL) for maternal toxicity and for embryo-foetotoxicity of β -myrcene administered orally (gavage) was reported to be 500 mg/kg bw/day in Wistar rats. This effect may however have arisen due to the significant maternal toxicity seen at 1200 mg/kg, rather than due to a direct effect of myrcene.

Data source

Reference
Reference Type:
publication
Title:
Study on Embryo-Foetotoxicity of beta-myrcene in the rat
Author:
Delgado, IF, Carvalho, RR, De Almeida Nogueira, ACM, Mattos, AP, Figueiredo, LH, Oliveira, SHP, Chahoud, I, and Paumgartten, FJR
Year:
1993
Bibliographic source:
Fd Chem Toxic Vol 31, no. 1, pp. 31-35, 1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
mating conditions (1 male for 3 females); food consumption not followed; bodyweight only recorded at Day 0, 6, 16 and 20 of gestation
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): beta-myrcene
- Molecular formula (if other than submission substance): C10H16
- Molecular weight (if other than submission substance): 136.24
- Smiles notation (if other than submission substance): See attached
- Structural formula attached as image file (if other than submission substance): see attached
Name of test material (as cited in study report): β-Myrcene
- Name of test material (as cited in study report): β-myrcene
- Source: Sigma Chemical Co., St. Louis, MO
- Analytical purity: 90%

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Oswaldo Cruz Foundation Central Animal House breeding stock
- Housing: Housed in plastic cages with wood shavings as bedding
- Diet (e.g. ad libitum): Nuvital diet for laboratory animals, Nuvilab Ltd, Curitiba, Brazil; ad libitum
- Water (e.g. ad libitum): Tap water; ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23 °C
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test material was dissolved in corn oil
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
None
Details on mating procedure:
Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:3
- Length of cohabitation: 2 h
- Proof of mating: Sperm in vaginal smear referred to as Day 0 of pregnancy
Duration of treatment / exposure:
10 days (Day 6-15 of pregnancy)
Frequency of treatment:
once daily
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 200 mg/kg bw/day
No. of animals per sex per dose:
Treated females: 22-36; pregnant females: 16-29
Control animals:
yes, concurrent no treatment
Details on study design:
Control animals: as no statistically significant difference was found between the untreated and the vehicle treated control animals, in any of the parameters analysed, the data of both groups were pooled and were thereafter designated the control group.

Examinations

Maternal examinations:
BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 16 and 20 of pregnancy
Ovaries and uterine content:
The uterine content was examined after termination: Yes, animals were sacrificed on Day 20 for caesarean examinations
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of living and dead foetuses: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:

- External examinations: Yes [all living foetuses]; weighed and examined for externally visible malformations
- Soft tissue examinations: Yes [5-7 litters per group]: evaluated for visceral malformations; heart, lungs, thymus, liver, spleen and kidneys were weighed and microdissected
- Skeletal examinations: Yes [all the remaining litters]
Statistics:

- Statistical analysis was by one way analysis of variance or, alternatively, by the Kruskal-Wallis test whenever the data did not fit a normal distribution
- Statistically significant differences between groups were tested by using a two sided Student's t-test or the Mann-Whitney U-test
- Proportions were analysed by the chi-square test
- Difference was considered to be statistically significant at P < 0.05
Indices:
na
Historical control data:
na

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: significant decreased body weight gain (1200 mg/kg)

Details on maternal toxic effects:
At 250 and 500 mg/kg bw /day: No treatment-related effects

At 1200 mg/kg bw/day:
- Mortality: 1/29 dams died on Day 11 of pregnancy
- Body weights: Statistically significant decrease in weight gain during days 6-11 of pregnancy (17.3 ± 7 g vs. 3.2 ± 13.2 g in control vs. 1200 mg/kg group, respectively)
- Cesarean examination: Number of visible implantation sites was significantly reduced (12.6 ± 2.2 vs. 10.2 ± 2.9 in control vs. 1200 mg/kg group, respectively)

Effect levels (maternal animals)

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

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:

- Body weight: Treatment-related effects on foetal weight were only very slight, or even insignificant, if the litter is taken as the statistical unit of analysis
- External examinations: Increased frequency of irregularly positioned hind paws was observed in 1200 mg/kg bw/day group
- Visceral examinations: Visceral malformations such as enlarged ureters associated with an enlarged renal pelvis (one control foetus and one in 1200 mg/kg group), shorter ureter (one control foetus and one in 250 mg/kg bw/day group) and accessory (seventh) lobe in the liver (three foetus in 250 mg/kg bw/day group)
- Skeletal examinations: Increased incidence of delayed ossification and minor gross structural anomalies in the foetal skeleton were observed in 1200 mg/kg bw/day group

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental toxicity

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: signs of retardation and of anomalies in the foetal skeleton observed at 1200 mg/kg bw/day

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
1 200 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Table 1. Occurrence of delayed ossification in foetuses of rats treated orally with β-myrcene on Days 6-15 of pregnancy

 

β-Myrcene (mg/kg bw)

Treatment

0

250

500

1200

Foetuses examined (no.)

114

106

116

209

Percentage of foetuses with signs of delayed ossificationαin:

Skull bones

4.4

0.9

3.4

9.6*

Caudal vertebrae

7

2.8

6

37.8*

Forelimbs/metacarpus

2.6

0

0.9

9.1*

Hind limbs/metatarsus

5.3

2.8

3.4

29.2*

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

* P < 0.05; chi-square test

Applicant's summary and conclusion

Conclusions:
In a read-across study, the no-observed-adverse-effect level (NOAEL) for maternal toxicity and for embryo-foetotoxicity of β-myrcene administered orally (gavage) was reported to be 500 mg/kg bw/day in Wistar rats.
Executive summary:

A study was conducted to evaluate the embryo-foetotoxic potential of β-myrcene in the Wistar rats similarly to OECD Guideline 414.

β -Myrcene (250, 500 and 1200 mg/kg bw/day) in corn oil was given orally to Wistar rats from Day 6 to 15 of pregnancy. Two control groups, one received vehicle only and another without treatment, were also studied simultaneously. All rats were weighed on Day 0, 6, 16 and 20 of pregnancy. Caesarean sections were performed on Day 20 of pregnancy, and the number of resorptions and implantation sites were recorded. Foetuses were weighed and examined for external, visceral and skeletal malformations.

A significant effect (decrease) on maternal body weight gain (~ 82%%) was seen during gestation days 6 to 11, at 1200 mg/kg. There was also an increased incidence of delayed foetal ossification in the 1200 mg/kg dose group. At lower dose levels no maternal or foetal effects were observed that were considered to be related to treatment. The possibility that the delay in skeletal ossification seen in foetuses at 1200 mg/kg may have been caused indirectly by the significant maternal toxicity cannot be precluded.

In conclusion, the no-observed-adverse-effect level (NOAEL) for maternal toxicity and for embryo-foetotoxicity of β -myrcene administered orally (gavage) was reported to be 500 mg/kg bw/day in Wistar rats. This effect may however have arisen due to the significant maternal toxicity seen at 1200 mg/kg, rather than due to a direct effect of myrcene.