Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 February 1992 to 13 March 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
The test animals were exposed to the test material orally from Day 6-15 of gestation instead of guideline recommendation of dosing from implantation (e.g., day 5 post mating) to the day prior to scheduled caesarean section.
GLP compliance:
yes
Remarks:
According to US FDA GLP Regulations
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
other: Charles River Crl: CD VAF/Plus
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 77 days old at the beginning of the study and approximately 12.5 weeks at the time of pairing
- Weight at study initiation: 225 - 296 g (on gestation day 0)
- Fasting period before study: Not reported
- Housing: The females were housed individually (except for first five days of acclimation; two/cages) in suspended, stainless steel, wire-mesh cages from receipt until euthanasia.
- Diet: Basal laboratory diet; ad libitum
- Water: Tap water; ad libitum
- Acclimation period: The rats were acclimated for a period of 11 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 22.2 - 23.3°C
- Relative Humidity: 55 ± 8%
- Air changes: Not reported
- Photoperiod: 12 hrs Fluorescent lighting/12 hrs dark

IN-LIFE DATES: From Feb. 07, 1992 to Mar.13, 1992

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was prepared for dosing as a solution in corn oil.
- Rate of preparation of dose formulation: Weekly
- Dose volume: 10 mL/kg bw

VEHICLE
- Concentration in vehicle: 10, 50 and 150 mg/mL for 100, 500 and 1500 mg/kg bw/day, respectively
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A sponsor supplied procedure was used in Week 1 which was later modified and included an extra step of the addition of methanolic extract to eliminate the presence of corn oil in final dilutions. This method was subsequently used for analysis of Week 2 and 10-day stability samples.
- Analysis of dosing suspensions was conducted weekly.

TEST SUBSTANCE STABILITY, HOMOGENEITY AND CONCENTRATION ANALYSIS:
The dose mixtures held for 10 days at room temperature contained 101 to 125% of test material concentration indicating stability. Mean homogeneity obtained for all the dose samples were below the target concentrations.
The test mixtures prepared on two separate occasions during the study contained 84 to 102% of the desired test material concentrations.
Details on mating procedure:
- Impregnation procedure: Cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: Until mating
- Verification of same strain and source of both sexes: Yes, the female and male animals were from the same source and strain.
- Proof of pregnancy: Mating was determined by observing copulatory plug; when confirmed, the same day was referred to as Day 0 of pregnancy.
Duration of treatment / exposure:
Gestation days 6 - 15
Frequency of treatment:
Once daily
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
30 inseminated females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosages were selected by a previous preliminary range finding study conducted separately. Dose levels of 125, 250, 500, 1000 and 3000 mg/kg bw/day of the test material were administered orally as a single daily dose on gestation days 6 through 15 at a volume of 10 mL/kg to mated Charles River Crl: CD female rats. Maternal toxicity was observed at 3000 mg/kg bw/day group. No decisive treatment related indications of developmental toxicity was observed at the tested dose levels. The NOAEL for maternal toxicity was determined to be 1000 mg/kg bw/day.
- Rationale for animal assignment: The mated females were assigned consecutively in a block design to one control and three test animals. The order in which the mated females were assigned corresponded to the day on which copulation was observed and the order in which the animals appeared in the breeding record.
- Rationale for test system: The rat strain used is appropriate for the study because it is susceptible to known developmental toxicants and the lab had historical control data.
- Rationale for route of administration: Oral route was considered to be the most closely equivalent to typical route of human exposure.

Examinations

Maternal examinations:
MORTALITY: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily
- Observations: Signs of toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each inseminated female rat was recorded on Days 0, 6, 9, 12, 16 and 20 of gestation.

FOOD CONSUMPTION: Recorded on the corresponding body weight days and was calculated for the following intervals: gestation days 0-6, 6-9, 9-12, 12-16, 16-20 and 0-20. Values for non-gravid animals were excluded from numerisation.

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20, by carbon dioxide inhalation
- Organs examined: Necropsy was conducted to examine congenital abnormalities and macroscopic pathological changes of the organs. The ovaries and uteri were examined.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Location of viable and non-viable foetuses
- Early and late resorptions
- Number of total implantations and corpora lutea
Fetal examinations:
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, half per litter. About half of all foetuses were fixed in Bouin's fixative for determination of visceral abnormalities (Wilson technique).
- Skeletal examinations: Yes, half per litter. About half of the foetuses from each litter were fixed in alcohol, macerated in potassium hydroxide and was stained with Alizarin Red S and cleared with glycerine for subsequent skeletal determination.
- Head examinations: No
Statistics:
Data were processed where appropriate to give group mean values and standard deviations. Statistical methods like t-test, ANOVA, Bartlett's test, Chi-square test, Fisher's exact test, Kruskal Wallis test and Mann Whitney-U-test with Bonferroni correction were used wherever appropriate.
Indices:
a) Percentage pre-implantation loss = ((Number of corpora lutea - Number of implantations) / Number of corpora lutea) × 100
b) Percentage post-implantation loss = ((Number of implantations - Number of viable foetuses) / Number of implantations) × 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group, 1500mg/kg bw/day, staining was observed in anogenital and/body surface; red or brown material around the nose, mouth and/or eyes; and/or staining around mouth. Post-dose salivation was observed for most animals in mid- and high- dose groups. The clinical signs in the high- dose group were attributed to the treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
One animal died on gestation Day 6, after dosing, in the group being dosed with 1500 mg/kg bw/day. The necropsy findings of this animal revealed no gross lesions. Another animal died on gestation Day 13 in the same group. Necropsy revealed discoloration of lungs. As the mortalities are observed only in this high dose group, it was attributed to the test material treatment. No mortalities were observed in the remaining groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A slight, but probably biologically significant, inhibition of maternal body weight gain during gestation days 6 - 9 and over the whole treatment interval (6 - 16) was observed for high dose group when compared with the control group. This was followed by marked body weight gain for the high dose group dams during gestation days 9-12 compared to the control dams and it was attributed to the treatment with the test material. Mean maternal body weights in all treatment groups and maternal body weight gains in low- and mid-dose group were generally comparable with the control group values throughout the study period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Significantly decreased treatment related reductions in food consumption were noted in the high- dose group during the treatment period. However, mean maternal food consumption was comparable in low-dose, mid-dose and control dams.
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:
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative liver weights increased significantly in high-dose group in comparison to the control group. This increase was attributed to test material.
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The percentage pre-implantation losses observed at 0 (control), 100, 500 and 1500 mg/kg bw/day were 11, 10.8, 8.7 and 6.7, respectively. The percentage post-implantation loss observed in control, 100, 500 and 1500 mg/kg bw/day groups were 4.5, 7.5, 4.3 and 7, respectively. It indicated that significantly higher early resorptions and/or post implantation loses were observed in 100 and 1500 mg/kg bw/day dose groups in comparison to the control groups. Since no other indications of the developmental toxicity were observed in any group, and since these differences from control group were small, they were attributed to normal biological variability rather than the test material.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of viable foetuses per dam was comparable between the control and treatment groups.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The pregnancy incidence observed at 0 (control), 100, 500 and 1500 mg/kg bw/day was 96.6, 93.3, 100 and 93.3 %, respectively.
Other effects:
not specified

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean foetal body weights in the treatment groups were comparable with the control group.
External malformations:
no effects observed
Description (incidence and severity):
No treatment related and biologically significant malformations were observed in the test material treated groups in comparison to the control group.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No treatment related and biologically significant malformations were observed in the test material treated groups in comparison to the control group.
Visceral malformations:
no effects observed
Description (incidence and severity):
No treatment related and biologically significant malformations were observed in the test material treated groups in comparison to the control group.
Other effects:
no effects observed
Description (incidence and severity):
No treatment related and biologically significant treatment-related developmental variations were observed in the test material treated groups in comparison to the control group.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
> 1 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at the highest (1500 mg/kg bw/day) dose level.
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Summary of caesarean data (Study # 37645)

Dose Group (mg/kg bw/day)

Total number of pregnancies

Percentage

Mean number of corpora lutea

Pre-implantation loss (%)

Post-implantation loss (%)

Mean early resorptions

Mean late resorptions

Control

29/30

96.66

18.8

11

4.5

0.7

0

100

28/30

93.33

18.2

10.8

7.5

1.2

0

500

30/30

100

17.8

8.7

4.3

0.7

0

1500

28/30

93.33

18.3

6.7

7

1.2

0

Applicant's summary and conclusion

Conclusions:
Under test conditions, administration of 3-L-Menthoxypropane-1,2-diol (Takasago Coolact 10) to female Crl: CD VAF/Plus rats during Day 6-15 of gestation, at dose levels of 100, 500 and 1500 mg/kg bw/day by intragastric intubation revealed a NOAEL of 500 mg/kg bw/day for maternal toxicity and NOAEL of >1500 mg/kg bw/day for developmental toxicity.
Executive summary:

The developmental toxicity study of 3-L-Menthoxypropane-1,2-diol (Takasago Coolact 10) was conducted following methods comparable to the OECD Guideline 414 (Prenatal Developmental Toxicity Study).

Sexually mature Female Charles River Crl: CD VAF/Plus rats weighing 225-296 grams were housed individually (except for first five days of acclimation; two/cages) in suspended, stainless steel, wire-mesh cages.

The animals were maintained under standard laboratory conditions (temperature: 22.2 - 23.3°C, humidity: 55 ± 8%, 12 h fluorescent light/12 h dark cycle per day) and fed on basal laboratory diet and given tap water, ad libitum. The female rats were cohoused with male rats (M/F ratio: 1:1, from same strain and supplier) for mating. Mating was determined by observing copulatory plug; and when confirmed, the same day was referred to as Day 0 of pregnancy.

The inseminated rats were divided into following groups: 0 (vehicle), 100 (low-dose), 500 (mid-dose) and 1500 (high-dose) mg/kg bw/day and were administered with test or control substances from Day 6 through Day 15 post conception. These dosages were selected by a previous preliminary range finding study conducted separately. Each group included 30 inseminated females. Prior to administration, the test material was dissolved in corn oil. Corn oil served as the vehicle control.

All animals were observed daily for mortality and clinical observations. The body weight of each inseminated female rat was recorded on Days 0, 6, 9, 12, 16 and 20 of gestation. Food consumption was recorded on the corresponding body weight days and was calculated for gestation days 0-6, 6-9, 9-12, 12-16, 16-20 and 0-20. Values for non-gravid animals were excluded from numerisation.

On gestation Day 20, the dams were euthanised by carbon dioxide inhalation and examined for congenital abnormalities and macroscopic pathological changes. The ovaries and uterine content (location of viable and non-viable foetuses, early and late resorptions and number of total implantations and corpora lutea) were examined after sacrifice.

Numbers of live and dead foetuses were determined. Each foetus was weighed, sexed and examined externally. About half of the foetuses from each litter were fixed in Bouin's fixative for determination of visceral abnormalities. The other half of foetuses from each litter were fixed in alcohol, macerated in potassium hydroxide and was stained with Alizarin Red S and cleared with glycerine for subsequent skeletal determination.

One animal died on gestation Day 6, after dosing, in treatment group 1500 mg/kg bw/day. The necropsy findings of this animal revealed no gross lesions. Another animal died on gestation Day 13 in the same group. Necropsy revealed discoloration of lungs. As the mortalities are observed only in the high dose group, it was attributed to the test material treatment. No mortalities were observed in the remaining groups.

In the 1500 mg/kg bw/day group, staining was observed in anogenital and/body surface; red or brown material around the nose, mouth and/or eyes; and/or staining around mouth. Post-dose salivation was observed for most animals in 500 and 1500 mg/kg bw/day dose groups. The clinical signs in 1500 mg/kg bw/day dose group were attributed to the treatment.

A slight, but probably biologically significant, inhibition of maternal body weight gain during gestation days 6-9 and over the whole treatment interval (6-16) was observed for 1500 mg/kg bw/day group when compared with the control group. This was followed by marked body weight gain for the high dose group dams during gestation days 9-12 compared to the control dams and it was attributed to the treatment with the test material.

Mean maternal body weights in all treatment groups and maternal body weight gains in 100 and 500 mg/kg bw/day dose group were generally comparable with the control group values throughout the study period. Significantly decreased treatment related reductions in food consumptions were noted in the 1500 mg/kg bw/day dose group during the treatment period. However, mean maternal food consumption was comparable in 100, 500 mg/kg bw/day and control dams.

In necropsy, absolute and relative liver weights increased significantly in 1500 mg/kg bw/day group in comparison to the control group. This increase was attributed to test material.

The pregnancy incidence observed at 0 (control), 100, 500 and 1500 mg/kg bw/day was 96.6, 93.3, 100 and 93.3 %, respectively. Significantly higher early resorptions and/or post implantation loses were observed in 100 and 1500 mg/kg bw/day dose groups in comparison to the control groups. Since no other indications of the developmental toxicity were observed in any group, and since these differences from control group were small, they were attributed to normal biological variability rather than the test substance.

The number of viable foetuses per dam was comparable between the control and treatment groups. Mean foetal body weights in the treatment groups were comparable with the control group.

No treatment related and biologically significant malformations and developmental variations were observed in the test substance treated groups in comparison to the control group.

Under test conditions, administration of 3-L-Menthoxypropane-1,2-diol ( Takasago Coolact 10) to female Crl: CD VAF/Plus rats during Day 6-15 of gestation, at dose levels of 100, 500 and 1500 mg/kg bw/day by intragastric intubation revealed a NOAEL of 500 mg/kg bw/day for maternal toxicity and NOAEL of > 1500 mg/kg bw/day for developmental toxicity.

This teratogenicity study is classified as acceptable, and satisfies the guideline requirements of OECD 414 method.