Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (SIDS documents; Iuclid reference [28]; Reliability assigned: 1)
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
2007
Report Date:
2007
Reference Type:
other: summary of toxicity studies
Title:
Unnamed
Year:
2007
Report Date:
2007
Reference Type:
secondary source
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Estrous cycle length and pattern, and anogenital distances were not performed because the test was conducted by the TG adopted in 1990.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Source: KYOWA HAKKO KOGYO CO., LTD.
- Analytical purity: 92.7 %
- Lot/batch No.: 70713

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: 10 weeks
- Weight at study initiation: 335-399g for males,204-260g for females

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Isononanol was dissolved in olive oil to give concentrations of 0.24, 1.2, and 6 % weight/volume.
New solutions were prepared every 7 days.


DIET PREPARATION
- Rate of preparation of diet (frequency): n.a.
- Mixing appropriate amounts with (Type of food): n.a.
- Storage temperature of food: n.a.


VEHICLE
- vehicle: olive oil
- Justification for use and choice of vehicle: test substance insoluble in water
- Concentration in vehicle: 0.24, 1.2, and 6 % weight/volume
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. Yakuhan Pharmaceutical Ltd.
- Purity: Japan Pharmacopoiea
Details on mating procedure:
no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The Japan Food Analytical Center (Foundation) confirmed that the prepared solution of the drug in various concentrations had the prescribed concentration and that it was uniform. No further technical details are reported.
Duration of treatment / exposure:
Males 46 days, females 14 days before mating to day 3 of lactation
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (vehicle), 12, 60, 300 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a range finding study; details cf. the study summary contained in section 7.5.1
Positive control:
Not required

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations were included


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): n.a.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): n.a.


OTHER:
- parental repeated toxicity: cf. Chapter 7.5.1, MHLW (1997)
Oestrous cyclicity (parental animals):
Yes
Sperm parameters (parental animals):
No
Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
- number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated were prepared for microscopic examination and weighed, respectively:
all animals: liver, kidney, spleen, heart, lung, brain, pituitary gland, thymus, adrenal, thyroid, stomach, small intestine, appendix, large
intestine, prostate gland and ovary, testes and epididymes (see also same study summary in section 7.5.1 for further details)
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:


GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]
Detail information not contained in secondary source ("Foetal:all macroscopic examination of all pups")
HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
etail information not contained in secondary source ("Foetal:all macroscopic examination of all pups")
Statistics:
Chi-square test (data on sexual cycling, copulation rate, conception rate, birthrate, nursing rate), Fisher's direct probability test, Bartlett's test, Kruskal-Wallis test, Dunnett's test or Mann-Whitney's U-test
Reproductive indices:
Copulation index: No. of pairs with succesful copulation/no. of pairs mated)x100
Implantation index: No. of implantation sites/no. of corpora lutea)x100
Live birth index: No. of live pups born/no. of pups born)x100
Offspring viability indices:
Live birth index: No. of live pups born/no. of pups born)x100
Viability index: No. of live pups on day 4/no.of live pups born)x100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
cross reference to the same study in Chapter 7.5.1


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
cross reference to the same study in Chapter 7.5.1


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS):
n.a., gavage study


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Estrous cycle length and pattern, and anogenital distances were not performed because the test was conducted by the TG adopted in 1990.



REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Not examined


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
As for the reproductive ability of parental animals, no effects were detected in any dose group in males.
In females prolongation of diestrous phase and decrease in implantation rate were observed in the 300 mg/kg group.
Total litter loss in two dams of the 300 mg/kg group was observed. The number of pups born alive were decreased in the 60 and 300 mg/kg groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
cross reference to the same study in Chapter 7.5.1

GROSS PATHOLOGY (PARENTAL ANIMALS)
cross reference to the same study in Chapter 7.5.1

HISTOPATHOLOGY (PARENTAL ANIMALS)
cross reference to the same study in Chapter 7.5.1

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: No effect at the highest tested dose
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: Prolongation of diestrous Decrease in implantation rate Total litter loss in 2/12 dams at 300 mg/kg bw/day

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not specified

Details on results (F1)

VIABILITY (OFFSPRING)
Viability on day 4 of lactation was decreased in the 300 mg/kg group male and female pups of the 300 mg/kg group showed lower body weights on day 0 of lactation.

CLINICAL SIGNS (OFFSPRING)
No data

BODY WEIGHT (OFFSPRING)
Male and female pups of the 300 mg/kg group showed lower body weights on day 0 of lactation.

SEXUAL MATURATION (OFFSPRING)
No data

ORGAN WEIGHTS (OFFSPRING)
No data

GROSS PATHOLOGY (OFFSPRING)
No findings

HISTOPATHOLOGY (OFFSPRING)
No data

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
12 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Number of live pups born / on day 4: reduced at 60 mg/kg bw/day Reduced implantation index at 60 mg/kg bw/day is considered to reflect embryotoxicity

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

As for the reproductive ability of parental animals, no effects were detected in males, but continuous diestrous was observed in females of the 300 mg/kg group on estrous cycle examination. All litters of animals in the 300 mg/kg group died, and implantation rate and number of pups born alive were decreased in the 60 and 300 mg/kg groups, along with numbers of implantations and pups born in the 300 mg/kg group. With regard to the effects on neonates, viability on day 4 of lactation was decreased in the 300 mg/kg group, and male and female pups of the 300 mg/kg group showed lower body weights on day 0 of lactation.

On the basis of these findings, NOAELs of 3,5,5-trimethylhexan-1-ol for reproductive/developmental toxicity were considered to be 300 mg/kg/day for males, 60 mg/kg/day for females, and 12 mg/kg/day for the F1 generation, respectively.

Detail Information:

Reproduction Toxicity parental animals

In sexual cycle findings for females, continuation of anestrus for 8 to 10 days was confirmed in four cases in the 300 mg/kg group. Although  copulation and pregnancy were successful in three of these cases, there was one case of unsuccessful copulation. Compared to the control group, there was no significant difference in the number of days to copulation, the copulation rate and the conception rate.

The number of implantations was decreased at 60 and 300 mg/kg bw; therefore, the implantation index was significantly reduced (p<0.05) at these dose levels. Maternal toxicity was also seen at 60 and 300 mg/kg bw (cf. summary of the same study in section 7.5.1).

Dose level (mg/kg bw/day)

0

12

60

300

No. of animals

12

12

12

12

No. of pregnant

11

11

12

11

No. of dead or sacrificed

0

0

0

4

No. of examined

11

11

12

7

Duration of mating (days, Mean±S.D.)

3.4±1.6

3.2±0.9

2.6±0.9

4.2±3.4

Copulation index (%) (a)

100.0

100.0

100.0

91.7

Fertility index (%) (b)

91.7

91.7

100.0

100.0

No. of corpora lutea

16.3±1.7

16.4±1.5

16.4±2.5

16.1±3.6

No. of implantation sites

15.8±1.7

15.0±1.5

14.3±1.8

13.4*±2.1

Implantation index (%) (c)

97.4±3.8

91.5±9.8

88.2* ±12.0

85.0*±14.5

Gestation length (days)

22.3±0.5

22.1±0.3

22.3±0.7

22.1±0.4

Gestation index (%) (d)

100.0

100.0

100.0

63.6

Values are expressed as Mean±S.D.

Significantly different from 0 mg/kg group; * p=0.05               **p=0.01

(a) (No. of pairs with succesful copulation/no. of pairs mated) x 100

(b) (No. of pregnant animals/No. of pairs with successful copulation) x 100

(c) (No. of implantation sites/no. of corpora lutea) x 100

(d) (No. of females with live pups delivered/No. of pregnant females) x 100

Fetal data

Compared to controls, low values for the number of nidations, nidation rate, number of births and the number of live births were seen at 60 mg/kg bw/day and in the 300 mg/kg/day group; a level of significance up to p<0.01 was gained for some of the parameters. Male and female pup weight at birth was reduced at 60 mg/kg bw/day and at 300 mg/kg bw/day (p<0.5 (males), p<0.01 (females)). Survival of pups until day 4 after birth was significantly reduced (p<0.01) in the group at 300 mg/kg bw/day.

No aberrations were confirmed in any of the cases in necropsies of deaths and newborn pups sacrificed on day 4 of nursing. 

Dose level (mg/kg bw/day)

0

12

60

300

Number of dams examined

11

11

12

7

No. of pups born

14.9±1.8

14.3±2.1

12.6±2.3

11.7±3.3*

Delivery index (%) (a)

94.1±5.4

95.3±4.2

88.1±13.2

87.5±19.1

No. of live pups born

14.8±1.7

14.2±2.0

12.5±2.4*

10.1±2.7**

Live birth index(%) (b)

99.4±1.9

99.5±1.7

99.2±2.6

88.9±18.4

Sex ratio (M/F)

1.29±0.48

1.12±0.69

0.81±0.37**

1.53±0.93

No. of dead pups born

0.1±0.3

0.1±0.3

0.1±0.3

1.6±2.9

Live pups on day 4

14.5±1.9

14.0±1.9

12.0±2.4*

6.6±5.1**

Viability index(%) (c)

98.0±3.4

98.8±2.6

96.3±8.4

64.9±44.6**

Body weight of pups (gr)

   Male, day 0

6.49±0.61

6.15±0.41

6.26±0.72

5.36±1.21*

   Male, day 4

10.55±1.02

9.58±0.68

10.51±1.60

10.22±1.75 (d)

   Male body weight gain (%) (e)

62.63±5.60

55.74±6.02**

67.55±13.40

71.59±8.50 (d)

   Female, day 0

6.25±0.74

5.94±0.47

5.91±0.63

5.03±1.21**

   Female, day 4

10.14±1.42

9.22±0.88

9.96±1.50

9.74±1.89 (d)

   Female body weight gain (%) (e)

61.97±5.41

55.36±9.63

68.08±12.64

73.22±9.89 (d)

Values are expressed as Mean±S.D.

Significantly different from 0 mg/kg group; * p=0.05               **p=0.01

(a) (No. of pups born/no. of implantations) x 100

(b) (No. of live pups born/no. of pups born) x 100

(c) (No. of live pups on day 4/no.of live pups born) x 100

(d) number of dams: 5

(e) (Body weight gain/body weight on day 0) x 100

Applicant's summary and conclusion

Conclusions:
Toxicity to reproduction, but no teratogenicity, was seen at 60 and 300 mg/kg bw /day. No toxicity to reproduction was seen in the absence of maternal toxicity. The NOAELs (reproduction) were males: 300 mg/kg bw/day.; females: 60 mg/kg bw/day; embryotoxicity: 12 mg/kg bw/day.
Executive summary:

3, ,5 ,5 -trimethylhexan-1-ol was tested in a combined repeated toxicity/reproduction toxicity screening test according to OECD TG 422 under GLP conditions in rats at 0, 12, 60, and 300 mg/kg bw/day (gavage). The repeated dose NOAEL was12 mg/kg bw/day in male and female parental animals (findings of this study are summarized in Chapter 7.5.1).

As to reproduction, the following deviations from normal were seen:

Continuation of anestrus for 8 to 14 days in 4 of the high dose females; pregnancy occurred in 3 of these.

The number of implantations was decreased at 60 and 300 mg/kg bw; therefore, the implantation index was significantly reduced (p<0.05) at these dose levels. Maternal toxicity was also seen at 60 and 300 mg/kg bw (cf. summary of the same study in section 7.5.1). Thus, compared to controls, low values for the number of nidations, nidation rate, number of births and the number of live births were seen at 60 mg/kg bw/day and in the 300 mg/kg/day group; a level of significance up to p<0.01 was gained for some of the parameters.

Male and female pup weight at birth was slightly reduced at 60 mg/kg bw/day, and statistically significantly reduced at 300 mg/kg bw/day (p<0.5 (males), p<0.01 (females)). Male and female body weights increased and were comparable with that of the controls on day 4 past parturition.

The viability index, i.e. survival of pups from birth until day 4 after birth, was significantly reduced in the group at 300 mg/kg bw/day (p<0.01). The authors related this to maternal toxicity during fetal development and during lactation.

No external aberrations were confirmed in any of the cases in necropsies of deaths and newborn pups sacrificed on day 4 of nursing. 

 

The authors derived the following NOAELs related to reproduction:

Males: 300 mg/kg bw/day; basis: no effect on virility

Females: 60 mg; basis: anestrus in 4 of 12 animals at high dose

Pups: 12 mg/kg bw/day; basis: nidation reduced, i.e. significantly reduced implantation index and lower number of fetuses at 60 mg/kg bw.

Overall, toxicity to reproduction was seen at maternal toxic dose levels (Yoshimura, 1997).

The study is considered to be suitable for assessment. It should, however, be noted that compared to the more specific guideline studies for this endpoint (e.g. OECD 416) the list of parameters to be examined is reduced. Further, the animal number is relatively low in screening studies and the statistical power is therefore reduced compared to an endpoint-specific OECD guideline study. The reduced implantation index seen at the 60 mg/kg dose level may therefore have failed to gain a level of statistical significance, making it difficult to assign correctly NOAEL-values.