Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an OECD guideline, GLP-compliant, reproductive screening study to rats (via the oral route) reproductive and developmental toxicity was assessed.

As an effect of test item on parent animals, death occurred at 30 mg/kg. In addition, decreases in body weights, food consumption and plasma total T4 concentration were observed at 30 mg/kg, and histopathological thickening of limiting ridge in the mucosa of the forestomach, which was suggestive of mucosal irritation of test item, was observed at 10 and 30 mg/kg.

Total litter loss was observed in 2 dams at 30 mg/kg on the day of delivery. Due to the total litter loss observed in the above animals, trends toward low values in gestation index and delivery index were observed at 30 mg/kg. In addition, trends toward low values in the number of live newborns and birth index, and trend toward a high value in the stillborn index were observed at 30 mg/kg.

In F1 male offspring, body weight was significantly lower on post-natal days (PNDs) 7 and 13 at 30 mg/kg than that in the control group. In F1 female offspring, body weight was significantly lower on PNDs 4, 7 and 13 at 30 mg/kg than that in the control group.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Adopted on 29 July 2016
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on species / strain selection:
This strain is widely used in reproduction/developmental toxicity studies using rodents, there is abundant historical data, and a large number of animals are available.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) ca. 10 wks
- Weight at study initiation: (P) Males: 362.2 - 418.3 g; Females: 217.9 - 258.4 g
- Housing: (1) For males and for females except the gestation and lactation periods; stainless hanging-type bracket cages (W × D × H: 226 × 346 × 198 mm), (2) For females during the gestation and lactation periods; polymethylpentene cages (W × D × H: 220 × 380 × 195 mm). Equipment for animal enrichment such as toys (alumina ball) and nesting materials and rest board was placed in the stainless cages. Nesting materials were placed in the polymethylpentene cages (including bedding). Toys and rest boards were exchanged once every two weeks or more frequently (6- to 14- day interval). Nesting materials were exchanged once a week or more frequently (1- to 7- day interval) when used in the stainless cages and concurrently with the cage exchange when used in the polymethylpentene cages.
- Diet (e.g. ad libitum) : ad libitum (rodent pellet diet)
- Water (e.g. ad libitum): ad libitum well water with sodium hypochlorite (free residual chlorine concentration: approx. 2 ppm)
- Acclimation period: 15 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24.1°C
- Humidity (%): 43.2 - 71.0%
- Air changes (per hr): 10-20 per hour
- Photoperiod (hrs dark / hrs light): 12h light per day (7:00 - 19:00)
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared under UV cut-off fluorescent light. The test substance was weighed in a glove box where air was replaced with nitrogen gas, and the relative humidity was confirmed to be below 30% with a hygrometer (actual value: 11% to 25%). The frequency of preparation of 1 and 3 mg/mL dosing formulations was once or twice every 6 or 7 days (permissible range: once or more every 8 days). The 0.3 mg/mL dosing formulation was prepared just before use. The vehicle (olive oil) was used as the dosing formulation for the control group.

(Control group dosing formulations)
The required amount of the vehicle was divided into polypropylene tubes for each dosing day.

(3 mg/mL dosing formulation)
(1) The test substance was weighed accurately and suspended by adding the vehicle gradually. Sonication (about 3 minutes) was repeated up to prepare a uniform suspension with an ultrasonic washing machine. Then the vehicle was added to make accurately the specified concentration (3 mg/mL) with a measuring cylinder.
(2) The dosing formulation after preparation was divided into polypropylene tubes for each dosing day.
(3) The tubes were filled with nitrogen gas.

0.3 and 1 mg/mL dosing formulations
(1) The 3 mg/mL dosing formulation was diluted with the vehicle to make 0.3 and 1 mg/mL dosing formulations. As for 0.3 mg/mL, the preparation was conducted before use.
(2) The dosing formulations after preparation were divided into polypropylene tubes for each dosing day.
(3) The tubes were filled with nitrogen gas.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation (GD0)
- After successful mating each pregnant female was caged (how): individually (1/litter)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance formulations at 0.4, 4, and 100 mg/mL were confirmed to be stable and homogenous under refrigeration, in a dark place, in a tube filled with nitrogen gas for 8 days in another study.

At the first preparation, the sampling dosing suspensions (n=3) from 3 points (upper, middle, and lower layers) of the 1 and 3 mg/mL dosing formulations were confirmed for concentrations (actual value: 99.4 and 101.7%, within ±10% of
the nominal concentration) and their homogeneity (actual value: 1.9 and 0.7%, C.V. of the upper, middle, and lower layers, within ±10%). The measurement was conducted in accordance with the analysis protocol. For the 0.3 mg/mL dosing formulation, the concentration of the test substance was confirmed by recording the actual amount of the test substance weighed and the final volume of the dosing formulation at each preparation. Homogeneity of the test substance was confirmed by visual inspection.

Method of analysis: Ion Chromatography
Duration of treatment / exposure:
Males: From 14 days before mating (Days 1 to 15) until the day before necropsy through the mating period (35 days in total).
Females: From 14 days before mating (Days 1 to 15) until Day 12 of lactation (day of delivery was Day 0 of lactation) through the mating and gestation periods and delivery. Non-mating females and non-delivery females were maintained until the day before necropsy.
Frequency of treatment:
Daily
Details on study schedule:
The day of the start of dosing was designated as Day 1, and Days 1 to 7 as Week 1. Days 1 to 15 were before the mating period. For females, the day of successful copulation was designated as Day 0 of gestation (GD 0) and the day of delivery as Day 0 of lactation (LD 0).
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (Vehicle)
Dose / conc.:
3 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Middle dose
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
12 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose level selection:
The dose levels were set based upon the results of a dose Range-Finding Study (28 Days Repeated Oral Administration in Rats). In this study, 1 of 5 males at 40 mg/kg died on Day 29 (scheduled necropsy day). In addition, decreased body weight and food consumption were observed in males at 40 mg/kg, and some abnormalities were observed at necropsy in both sexes at 40 mg/kg. Therefore, the high dose level was set at 30 mg/kg which was less likely to die and was expected to develop some toxicity. The middle and low dose levels were set at 10 and 3 mg/kg/day, respectively, at a geometric ratio of about 3.

- Rationale for animal assignment (if not random):
On the day before the start of administration for both sexes, 9 females showing 5-day or more estrous cycles were excluded from the group assignment on the basis of the results of estrous cycle examination. The other animals were assigned to each group by the stratified randomization on the basis of the body weights (computer system; Provantis®, Instem LSS Limited). The animals weighing within ± 20% of the mean body weights (calculated for each sex) were used for this study.

Positive control:
Not applicable
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed twice a day (before and after dosing) during the dosing period, and once a day in the other periods.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed on Days 1, 8, 15, 22, 29, and 36. Females were weighed on Days 1, 8, 15, and/or 22 during the pre-mating period, GDs 0, 7, 14, and 20 during the gestation period, and LDs 0, 4, 7, and 13 during the lactation period. One female in which cohabiting was discontinued on Day 19 due to death of the partner male (hereinafter referred to as non-mating female) was also weighed on Days 22, 29, and 36.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OBSERVATIONS DURING DELIVERY AND LACTATION:
- All females with successful copulation were allowed natural delivery. The observation of delivery was conducted once daily (a.m. 9:00) from GDs 21 to 24. Females that
delivered their litter completely by 9:00 a.m. were judged as “delivered” on the corresponding day (the delivery day was regarded as LD 0), and when delivery was
completed at 9:00 or later, the following day was defined as LD 0.

HORMONE CONCENTRATION (TOTAL T4) ANALYSIS
- For parental animals, total T4 concentration was measured for males. Since changes attributable to the test substance treatment was observed in males at 30 mg/kg, females (LD13) were subjected to additional measurement.

Oestrous cyclicity (parental animals):
Vaginal smears were collected with swabs from all females in the morning (approximately same time every day) from the day of the start of dosing to the day of confirmed copulation. The obtained smears were collected on a plate for each animal, and stained with Giemsa. The estrous cycle was classified into diestrus (D), proestrus (P), estrus (E), and metestrus (M). The mean estrous cycle (number of days from the estrous period to the next estrous period) and the number of the estrous period during the test period were calculated.
Sperm parameters (parental animals):
Parameters examined male parental generations:
- testis weight, epididymis weight, sperm count in epididymides
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter) random removal of F1 offspring

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

HORMONE CONCENTRATION (TOTAL T4) ANALYSIS
The offspring were subjected to the measurement on PND 13 only. No measurements were conducted on PND 4 in any offspring.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on Day 36
- Maternal animals: All surviving animals on Lactation Day 13

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations thoracoabdominal organs/tissues were examined macroscopically.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring on post natal day (PND) 13.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations thoracoabdominal organs/tissues were examined macroscopically.
Statistics:
Statistical analysis (SA) was performed for the following data, except for the sex ratio, using a computer system (Provantis®, Instem LSS Limited). SAS 9.4 (SAS Institute Japan [CAC Croit Corporation]) was used for the sex ratio. In any case, two-tailed test was used, and levels of p<0.01 and p<0.05 were judged significant. SA of hormone concentration (total T4) was performed at the test site 2 using SAS 9.4 (EXSUS Version 8.1.0, SAS Institute Japan Ltd.). The data of offspring were analyzed on the basis of litter mean values. Moreover, the body weight and food consumption of non-pregnant female as well as the body weight of non-mating female on and after the start of mating were excluded from the evaluation.
Concerning dams with total litter loss, SA was conducted on the body weight and food consumption up to the day before total litter loss.

Multiple comparison test
The mean and SD were calculated and homogeneity of variance was tested by Bartlett’s method (p<0.05). When the groups were accepted as homogeneous, Dunnett’s multiple comparison test was used for comparison of the groups of data. When the groups of data were found to be heterogeneous by Bartlett’s test, Steel’s multiple comparison test was conducted.
Items: Body weights, food consumption, absolute and relative organ weights, estrous cycle, number of estrus, days until copulation, gestation length, number of implantations, number of delivered offspring, number of live offspring, body weight of offspring (both sexes) and AGD

Chi-square test
chi-square test was performed between the control and test item-treated groups.
Items: Copulation index, fertility index, gestation index, delivery index, and sex ratio

Wilcoxon’s rank sum test
Wilcoxon’s rank sum test was performed between the control and test item treated groups.
Items: Stillborn index, external anomaly index, external anomaly typing index, live birth index, viability index on Day 4, viability index on Day 13, and nipple development anomaly index
Reproductive indices:
Gestation index (%): (Number of pregnant animals delivered with live offspring / number of pregnant animals) × 100
Delivery index (%): (Total number of offspring at birth / number of implantations) × 100
Offspring viability indices:
Birth index (%): (Number of live offspring at birth / number of implantations) × 100
Stillborn index (%): (Number of stillborns / total number of delivered offspring) × 100
Viability index on Day 4 (%): (Number of live offspring on PND 4 / number of live offspring at birth) × 100
Viability index on Day 13 (%): (Number of live offspring on PND 13 / number of live offspring after culling) × 100
Sex ratio: (Number of male live offspring / number of female live offspring)
External anomaly index (%): (Number of offspring with external anomaly / number of observed offspring) × 100
External anomaly typing index (%): (Number of offspring with external anomaly by each type / number of observed offspring) × 100
Nipple development anomaly index (%): (Number of offspring with nipple development anomaly / number of observed offspring) × 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Two males at 30 mg/kg died on Days 21 and 19, respectively. For these animals, decrease in locomotor activity, perioral smudge, smudge of perinasal area, or bradypnea was observed from Day 14 until the day of death.
Three dams at 30 mg/kg died from GD 23 to LD 2. In one of these dams, salivation was observed on LD 0. In the other dams, no abnormal clinical signs were observed until the day of death.
In the surviving animals, salivation was sporadically observed after dosing in 6 males at 30 mg/kg on Day 12 or later and 3 females on Day 15 or later.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Two males at 30 mg/kg died on Days 21 and 19, respectively.
Three dams at 30 mg/kg died from GD 23 to LD 2.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males, body weight was significantly lower at 30 mg/kg than that in the control group on Days 15, 29, and 36.
In females, body weight was significantly lower at 30 mg/kg than that in the control group on LDs 4 and 7.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males, food consumption was significantly lower at 30 mg/kg than that in the control group on Day 2.
In females, food consumption was significantly lower at 30 mg/kg than that in the control group on LDs 1 and 4.
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, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
[Dead animals (2 males and 3 females at 30 mg/kg)]
In males, erosion in the mucosa of the glandular stomach was observed in 2 animals.
In females, erosion in the mucosa of the forestomach, erosion in the mucosa of the glandular stomach, and hyperplasia of squamous epithelium in the limiting ridge of the stomach were observed in 1, 2, and 3 animals, respectively.

[At the end of administration period]
In males, hyperplasia of squamous epithelium in the limiting ridge of the stomach was observed in 2 and 6 animals at 10 and 30 mg/kg, respectively.

Focal lymphocytic infiltration in the epididymis was observed in 3 and 1 animals of the control and 30 mg/kg groups, respectively; however, this change was not considered to be treatment-related because there was no dose-dependency. Atrophy of seminiferous tubule in the testis and decreased sperm in the lumen of the epididymis were observed in one male with small testis and epididymis observed at necropsy in the control group.

In females, hyperplasia of squamous epithelium in the limiting ridge of the stomach was observed in 1 animal at 30 mg/kg.

[Total litter loss (2 females at 30 mg/kg)]
Two females with litter loss showed erosion in the mucosa of the glandular stomach. One female also showed hyperplasia of squamous epithelium in the limiting ridge of the stomach.

[Non-mating and non-pregnant females]
No abnormalities were observed in any female.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
HORMONE CONCENTRATION (TOTAl T4) ANALYSIS
In males (F0), plasma total T4 concentration was significantly decreased at 30 mg/kg than that in the control group. The plasma total T4 concentration of the test item treated groups (Groups low-, middle-, and high-dose) was equivalent to the control group in parental animals [F0, female (LD 13)] and offspring [F1, PND 13].
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed in estrus count or estrous cycle in any test item-treated group.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
testis weight, epididymis weight - no statistical significant difference between teratment groups and controls.
sperm count in the lumen epididymides - no effects on sperm count in treatment groups.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
No statistically significant differences were observed in the days until copulation, copulation index, or fertility index in any test item treated group. One female at 3 mg/kg did not become pregnant.
Accordingly, the copulation indices were 100% for the control and all test item-treated groups, and the fertility indices were 100%, 91.7%, 100%, and 100% for the control, 3, 10, and 30 mg/kg groups, respectively.

The total litter loss was observed in 2 dams at 30 mg/kg on LD 0. Their litter was all stillborn. Due to total litter loss, although there was no statistical significance, gestation index (77.8%) and delivery index (86.669%) were lower at 30 mg/kg than those in the control group. These values were outside the range of historical data at the test facility.

Historical data (2013 to 2016, 6 studies, 10 to 12 dams/study)
Gestation index Mean: 100%
Delivery index Mean: 91.868% to 94.255%

No statistically significant difference was observed in the gestation length or number of implantation between the control group and any test item treated group.

As an effect of test item on parent animals, death occurred at 30 mg/kg. In addition, decreases in body weights, food consumption and plasma total T4 concentration were observed at 30 mg/kg, and histopathological thickening of limiting ridge in the mucosa of the forestomach, which was suggestive of mucosal irritation of test item, was observed at 10 and 30 mg/kg.

Total litter loss was observed in 2 dams at 30 mg/kg on the day of delivery. Due to the total litter loss observed in the above animals, trends toward low values in gestation index and delivery index were observed at 30 mg/kg. In addition, trends toward low values in the number of live newborns and birth index, and trend toward a high value in the stillborn index were observed at 30 mg/kg.
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Total litter loss observed at 30 mg/kg in 2 dams
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
External examination of offspring (PND 13): No abnormalities were observed in any offspring. Offspring with external anomalies were observed in 1 offspring (0.649%) at 3 mg/kg. With regard to individual types of anomalies, acaudate was observed in 1 offspring (0.649%) at 3 mg/kg; however, this change was not considered to be treatment-related because there was no dose-dependency.
AGD (Anogenital distance, PND 4): No statistically significant difference was observed in the AGD between the control group and any test item -treated group.
Nipple development (PND 12): No statistically significant difference was observed in nipple development anomaly index between the control group and any test item-treated group.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The total litter loss was observed in 2 dams at 30 mg/kg on LD 0. Their litter was all stillborn.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In male offspring, body weight was significantly lower on PNDs 7 and 13 at 30 mg/kg than that in the control group. In female offspring, body weight was significantly lower on PNDs 4, 7 and 13 at 30 mg/kg than that in the control group.
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
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
HORMONE CONCENTRATION (TOTAL T4) ANALYSIS
The plasma total T4 concentration of the test item treated groups (Groups low-mid and high) for offspring [F1, PND 13] - no treatment related effects.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Although there was no statistical significance, the number of live newborns (9.4) and birth index (74.103%) were lower, and stillborn index (22.222%) was higher at 30 mg/kg than those in the control group. These values were outside the range of historical data at
the test facility.
Historical data (2013 to 2016, 6 studies, 10 to 12 dams/study)
Number of live newborns Mean: 12.8 to 14.6
Birth index Mean: 90.049% to 93.702%
Stillborn index Mean: 0% to 1.818%

No statistically significant difference was observed in the number of litter, sex ratio, or viability index on Day 4 or 13 between the control group and any test item-treated group.
Offspring with external anomalies were observed in 1 offspring (0.649%) at 3 mg/kg.
With regard to individual types of anomalies, acaudate was observed in 1 offspring (0.649%) at 3 mg/kg; however, this change was not considered to be treatment-related because there was no dose-dependency.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
body weight and weight gain
Reproductive effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
yes

Table 2: Gross pathology findings (P0)

Sex

Male

Female

Female

Dose (mg/kg)

30

30

30

Number of Animals

2

3

2

Reason for necropsy

Dead

Dead

Total Litter loss

Necropsy findings

 

Present

2

3

2

Stomach

 

Dark reddish change; mucosa, glandular stomach

2

0

0

Dark reddish patch; mucosa, glandular stomach

0

1

2

Thickening; limiting ridge, mucosa, forestomach

0

3

1

Abnormal contents; dark red

2

0

0

Duodenum

 

Abnormal contents; dark red

2

-

-

Jejunum

 

Abnormal contents; dark red

2

-

-

Ileum

 

Abnormal contents; dark red

1

-

-

Cecum

 

Abnormal contents; dark red

1

-

-

Table 3: Body weights (F1, lactation period)

Day(s) Relative to Littering (PND)

Control

3 mg/kg

10 mg/kg

30 mg/kg

Sum of pup count (female)

0

 

80

71

87

44

4

 

80

70

85

37

7

 

49

44

50

23

13

 

49

44

50

23

Mean female pup BW/Litter

0

Mean

6.703

6.627

6.777

6.370

SD

0.745

0.631

1.035

1.151

N

12

11

12

7

4

Mean

11.182

11.264

10.853

9.241*

SD

1.478

1.401

1.703

1.556

N

12

11

12

6

7

Mean

17.991

18.380

17.614

13.757**

SD

1.834

1.972

1.841

3.010

N

12

11

12

6

13

Mean

34.099

34.448

33.103

27.736**

SD

2.026

2.376

2.141

4.699

N

12

11

12

6

Sum of pup count (male)

0

 

72

69

67

41

4

 

72

69

67

39

7

 

47

43

44

25

13

 

47

43

44

25

Mean male pup BW/Litter

0

Mean

7.062

7.000

7.189

6.851

SD

0.762

0.789

1.001

1.177

N

12

11

12

7

4

Mean

11.737

11.789

11.494

9.918

SD

1.398

1.518

1.789

2.059

N

12

11

12

6

7

Mean

19.037

18.524

18.524

14.701*

SD

1.729

1.967

1.967

3.199

N

12

12

12

6

13

Mean

35.694

34.549

34.549

29.208*

SD

2.062

2.396

2.398

4.643

N

12

12

12

6

* d-test: Dunnett 2-sided p<0.05

** d-test: Dunnett 2-sided p<0.01

Table 4: mean plasma total T4 hormone measurements

Group

Dose (mg/kg day)

Total T4 (nmol/L)

P0, male

P0, female

F1, PND13

Control

0

40.4 ± 3.4

22.8 ± 4.8

47.6 ± 5.1

Low dose

3

39.4 ± 5.2

20.2 ± 2.9

50.6 ± 5.5

Middle dose

10

38.6 ± 5.8

24.1 ± 5.7

49.9 ± 6.7

High dose

30

34.5 ± 7.1*

23.7 ± 4.6

44.7 ± 12.4

* P<0.05: Significantly different from control group

Conclusions:
The NOAELs for this study were 10 mg/kg/day (reproductive and developmental toxicity).
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
All toxicological studies on the test substance have been conducted in accordance to OECD (or equivalent) guideline and GLP-compliant. Therefore the quality of the database is regarded as high quality.
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

In an OECD guideline, GLP-compliant, pre-natal developmental study peformed on rats (via the oral route) developmental toxicity was assessed.


Death occurred in 1 dam in the 30 mg/kg group on GD 20. This animal showed no
abnormalites that directly led to the death in clinical signs, body weights, or necropsy although low food consumption was noted; thus, the direct cause of death was unknown. However, the cause of death was judged to be effects of the substance treatment by the following reasons:
(1) the surviving dams in the 30 mg/kg group showed worsening of general
conditions and lower body weight and food consumption and
(2) deaths occurred at 30 mg/kg in the end of the gestation period in reproduction/developmental toxicity screening test of the substance.


In the surviving dams, effects of the substance treatment were observed in the 30 mg/kg group as follows. In the clinical signs, decrease in locomotor activity was observed in 1 dam from GDs 19 to 20 and soiled fur (chest or abdomen) in 2 dams on GD 20. Decreased body weight and food consumption were observed in the late phase of the gestation period (GD 15 or later). No effects of the substance treatment were observed in necropsy including gravid uterus weights,organ weights, or histopathological examination (thyroid gland), or plasma hormone concentration (total T3, total T4, or TSH) at any dose level.


No effects of the test substance were observed in examination of uterine contents (number of corpora lutea, implantations, live fetuses, or resorption, pre- implantation loss, or postimplantation loss) or examination of fetuses (sex ratio, fetal weights, AGD, or visceral, skeletal, external, or placental development).
Therefore, the substance is considered to have had no embryo-fetal lethality, growth retardation, or structural abnormalities in rats in this study.


In conclusion, the NOAEL of the test substance was considered to be 10 mg/kg for general toxicity and reproductive function in dams, and 30 mg/kg for embryonic/fetal development under the conditions of this study.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted 25 June, 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: Mating was started at 12 weeks of age by housing females with males on a one-to-one basis day and night.
- Weight at study initiation: Females 10 weeks 201.8 to 237.2 g and 11 weeks 214.3 to 251 g.
- Fasting period before study: None
- Housing: Hanging-type stainless wire mesh cages (W226 × D346 × H198 mm).
Quarantine and acclimation periods: 2 to 3 animals/cage, same sex
Mating period: 1 male and 1 female/cage
After group assignment: 1 animal/cage
After mating period: 1 male/cage
Remaining animals: 2 to 3 animals/cage
- Diet (e.g. ad libitum): Pellet diet for experimental animals available ad libitum
- Water (e.g. ad libitum): Well water provided ad libitum and analysed twice yearly.
- Acclimation period: The quarantine and acclimation periods were set from animal receipt to the day of the start of mating, including the quarantine period for 6 days after receipt.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.3°C to 24.6°C
- Humidity (%): 44.2% to 64.0%
- Air changes (per hr): 10 to 20 times per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycles

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing formulations were prepared under UV cut-off fluorescent light. The test substance was weighed in the glove box where air was replaced with nitrogen gas, and the relative humidity was confirmed to be below 30% with a hygrometer (actual value: 7% to 11%). The frequency of preparation of 1 and 3 mg/mL dosing formulations was prepared
once every 3 to 7 days (permissible range: once or more every 8 days). The 0.3 mg/mL dosing formulation was prepared just before use. The vehicle (olive oil) was used as the dosing formulation for the control group. The dosing formulations after preparation were divided into polypropylene tubes for each dosing day and filled with nitrogen gas.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Standard vehicle
- Concentration in vehicle: 0, 0.3, 1 and 3 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity: The test substance formulations at 0.4, 4, and 100 mg/mL were confirmed to be stable and homogenous under refrigeration, in a dark place, and in a tube filled with nitrogen gas for 8 days in another study.

Determination of test substance concentration and homogeneity in dosing formulations: Determination of the test substance concentration and homogeneity in the 0.3, 1 and 3 mg/mL dosing formulations were confirmed utilising ion chromatography as follows:

Ion chromatography system: ICS-1000 (No.1 DIONEX)
Workstation: Chromeleon
Autosampler: AS50
Column: DIONEX IonPac, AS14A, 4 mm i.d. x 250 mm
Guard column: DIONEX IonPac, AS14A, 4 mm i.d. x 50 mm
Column temperature: 35°C
Mobile phase: 8.0 mM NA2CO3/1.0 mM NaHCO3 (aqueous solution)
Flow rate: 1.2 mL/minute
Injection volume: 25 µL
Suppressor: AERS500 4mm

The test substance were adequately determined in dose formulations at 1 and 3 mg/mL (homogeneity also assessed at upper, middle and lower layers). The actual concentration was analysed as 100% at 1 mg/L and 98% at 3 mg/mL. The homogeneity values were within 10% across the layers, indicating successful formulation procedures were employed.

In the analysis of test substance concentration and homogeneity in the dosing formulations performed at the test site the analysis results of the initially prepared dosing formulations had failed to meet the criteria specified in the study protocol (concentration: within 100 ± 10%, homogeneity: CV value within 10%). The cause was insufficient stirring during analysis. Since the analysis results obtained using newly prepared dosing formulation in re-analysis conducted under sufficient stirring met the criteria and re-prepared dosing formulations were used for administration, it was judged that this incident had no effect on the reliability of the study.
Details on mating procedure:
Mating was started at 12 weeks of age by housing females with males on a one-to-one basis day and night. Successful copulation was confirmed by the presence of a vaginal plug or sperm in a vaginal smear taken in the morning; the day of confirmed copulation was designated as Day 0 of gestation (GD 0).
Duration of treatment / exposure:
The dosing formulations were administered to females for which copulation was confirmed once daily from GD 6 to GD 19 (for 14 days).
Frequency of treatment:
Once daily
Duration of test:
GD 6 to GD 19 (14 days dosing).
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
3 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Middle dose
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
20 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were set based on the results of a reproductive/developmental screening assay in rats (dose level: 0, 3, 10, and 30 mg/kg/day, vehicle: olive oil).
In this study, 3 of 12 dams at 30 mg/kg died from Day 23 of gestation to Day 2 of lactation. For these dams, necropsy and histopathological examination results showed abnormalities in the stomach. In addition, trends toward reduced newborns viability were observed at 30 mg/kg. Therefore, the high dose level was set at 30 mg/kg which was expected to develop some toxicity. The middle and low dose levels were set at 10 and 3 mg/kg, respectively, with a common ratio of about 3. A control group (0 mg/kg) dosed with the vehicle (olive oil) alone was also established.
- Rationale for animal assignment (if not random): Randomly assigned
- Fasting period before blood sampling for (rat) dam thyroid hormones: Animals not fasted.
- Time of day for (rat) dam blood sampling: GD 20 at scheduled necropsy. Timings for necropsy and blood sampling were not reported.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical signs and mortality were observed twice a day (before dosing and after dosing) during the dosing period and once a day during the other periods.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were measured on GDs 0, 3, 6, 9, 12, 15, 18, and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Food consumption was weighed on GDs 0 to 1, 2 to 3, 5 to 6, 8 to 9, 11 to 12, 14 to 15, 17 to 18, and 19 to 20. Feeders containing diet were weighed and set in the animal cages in the morning. On the following morning, the feeders were weighed to calculate the food consumption for each day. The day for food consumption was expressed on GDs 0, 2, 5, 8, 11, 14, 17, and 19.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The thoracoabdominal organs and tissues were immediately examined macroscopically after excision of the ovary and uterus. The thyroid (including parathyroid) was fixed in 10 vol% phosphate buffered formalin solution and preserved.

ORGAN WEIGHTS: Yes
After necropsy, the thyroid (including parathyroid) from all animals (except for a dead dam) were weighed (absolute weight) and the ratio of organ weight to body weight (relative weight) was calculated on the basis of body weight measured on the day of necropsy. Paired organs were measured together.

MICROSCOPIC EXAMINATION: Yes
The thyroid (including parathyroid, unilateral) from all dams was embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and examined microscopically. As a result, examination could not be conducted in 2 dams. A
non-pregnant animal (No. 539) was not examined.

While preparing parathyroid tissue specimens from 2 animals (Nos. 515 and 522) of the control and 3 mg/kg groups, respectively, the tissue side could not be placed on the paraffin section and therefore, histological examination could not be performed on that specimen. Because toxicological assessment was possible in parathyroids of the remaining animals (18 or 19 animals) in the same group, this incident had no effect on the reliability of the study.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

After the uterus weight (including the uterine cervix) was measured, the numbers of corpora lutea, implantations, early resorptions, late resorptions, dead fetuses, and live fetuses were counted and the placentas were observed macroscopically. The live fetuses were individually sexed, observed macroscopically for external anomalies, and weighed for body weights. The live fetuses were euthanized by overdose with thiopental sodium via an intraperitoneal injection. Fetuses allocated for skeletal examination were examined for the internal reproductive organs after euthanasia for confirmation of the position and morphology, as well as for their gender for the second time. As a result, no abnormality was found in the position or morphology of the internal reproductive organs, and the results of gender check corresponded to those obtained in the observation of external reproductive organs.

The uterus of 1 female (No. 539; 3 mg/kg group), in which implantation was not confirmed macroscopically was immersed in 10 vol% ammonium sulfide solution to detect implantation. As a result, this animal was determined to be nonpregnant.

Early resorptions, late resorptions, and dead fetuses were classified based on the following criteria:
Early resorption: Implantation site, placental remnant, and formless embryo.
Late resorption: Showing the form of a fetus, but with remarkable maceration due
to time lapse after death.
Dead fetus: Showing a similar development as a live fetus and with no maceration.
Blood sampling:
For all pregnant animals, the blood was sampled upon necropsy (GD 20).

The animals were anesthetized by tail vein injection of thiopental sodium (30 mg/kg). Approximately 2 mL of blood was collected into a syringe containing heparin sodium from the posterior vena cava. The blood was transferred to a polypropylene tube, immediately cooled on ice, and then promptly centrifuged (approximately 1870 × g, 10 min, approximately 4°C) to obtain plasma (500 μL or more). The collected plasma was stored frozen in a deep freezer (actual range: -81.2°C to -78.1°C, permissible range: -90°C to -65°C).

The total Triiodothyronine (T3), total Thyroxine (T4), and Thyroid-Stimulating Hormone (TSH) plasma concentrations were measured for pregnant animals.

All values of the QC samples were within the range of 100 +/- 25% of nominal value, and variations in duplicate cpm of the standard solutions were within the acceptable range. There were no abnormalities in the procedures for the determination or in the values of the test samples. Therefore, the plasma hormone concentration measurement results were judged to be reliable.
Fetal examinations:
Live fetuses were numbered in order, starting from fetuses near the right and left ovary. Fetuses were allocated, in each sex of each litter, as specimens for skeletal and visceral examinations. Approximately one half of live fetuses in each sex (as specimens for skeletal examination) of each litter was individually identified by tattooing their four limbs and fixed in 70 vol% ethanol. The other half of fetuses (as specimens for visceral examination) were individually identified by a dorsal number inscribed with an oil-based felt pen and fixed in a Bouin’s solution.

- External examinations: Yes: The live fetuses were individually sexed, observed macroscopically for external anomalies, and weighed for body weights.
- Soft tissue examinations: Yes: The fetuses in the control and high dose groups were examined. Fetuses fixed in Bouin’s solution were observed for visceral anomalies by using the razor blade section method of Wilson for the head, neck, and abdomen, and by the microdissection method of Nishimura for the chest. Gender was checked for the second time at observation of the internal reproductive organs. For the low and middle dose groups, only the gender check (observation of internal reproductive organs) was performed. As a result, the results of gender check corresponded to those obtained in the observation of external reproductive organs in all fetuses.
- Skeletal examinations: Yes: The fetuses in the control and high dose groups were examined. Fetuses fixed in 70% ethanol were stained with alizarin red S according to the method of Staples et al. and were observed with a stereomicroscope for skeletal anomalies, variations, and ossification progress. In the ossification progress, the numbers of sternebrae and sacrocaudal vertebral body were regarded as end-points. After skeletal examination was completed, all the skeletal specimens were preserved in 100 vol% glycerin containing thymol.
- Head examinations: Not examined
- Anogenital distance of all live rodent pups: The anogenital distance (AGD: distance between the anus and genital node) was measured for all live fetuses with a micrometer caliper (Mitutoyo). In order to correct variations in measured values due to weight differences among individuals, the AGI (anogenital index)
divided by the cubic root of the body weight on the measurement day was also calculated.
Statistics:
Statistical analysis was performed with a computer system (tsPharma LabSite). As to the sex ratio, SAS 9.4 (SAS Institute Japan [CAC Croit Corporation]) was used. In any case, two-tailed test was used and levels of p<0.01 and p<0.05 were considered significant.

The body weight and food consumption of the non-pregnant female were excluded from the evaluation. Concerning the dead dam, statistical analysis was conducted on the body weight and food consumption while the animal was alive.
Indices:
Sex ratio of live fetuses: Number of live male fetuses / number of live male and female fetuses

Pre-implantation loss index, Post-implantation loss index, Early resorption index, Late resorption index, Dead fetus index, Incidence of fetuses with external anomalies (by type of anomaly), Incidence of fetuses with placental anomalies (by type of anomaly), Incidence of fetuses with skeletal anomalies (by type of anomaly), Incidence of fetuses with skeletal variations (by type of variation) and Incidence of fetuses with visceral anomalies (by type of anomaly).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the surviving animals, a decrease in locomotor activity was observed in 1 dam (No. 564) in the 30 mg/kg group from GDs 19 to 20 and soiled fur (chest or abdomen) was observed in 2 dams (Nos. 564 and 577) in the 30 mg/kg group on GD 20.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
In the 30 mg/kg group, 1 dam (No. 572) was found dead on GD 20. In the
clinical observation, no remarkable change was observed in this animal throughout the experimental period. The food consumption was low (0.4 g) at death. In the body weights, necropsy, and histopathological examination of the thyroid, no abnormalities were observed in this animal.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight in the 30 mg/kg group was significantly lower when compared with the control group on GD 20. Individually in the 30 mg/kg group, a continuous decrease in body weight was observed in 1 dam in the mid to late stages of gestation (No. 564: -13.4 g from GDs 15 to 18 and -23.6 g from GDs 18 to 20) and a decrease in body weight was observed in 4 dams in the late stage of gestation (No. 565 : -14.2 g, No.567 : -7.0 g, No.577 : -38.3 g, No. 580: -1.6 g from GDs 18 to 20).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption in the 30 mg/kg group was significantly lower when compared with the control group from GDs 19 to 20. Individually in the 30 mg/kg group, an apparent decrease in food consumption was continuously observed in 1 dam in the late stage of gestation (No. 564: 2.5 g from GDs 17 to 18; 1.2 g from GDs 19 to 20) and was observed in 5 dams (No. 565: 2.5 g, No. 567: 7.1 g, No. 577: 0.1 g, No. 579: 9.0 g, No. 580: 4.0 g from GDs 19 to 20).
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
Endocrine findings:
no effects observed
Description (incidence and severity):
No statistically significant differences were observed in the total T3, total T4, and TSH between the control and any test substance treatment group.

Tabulated results can be found in the section 'Any other information on results incl. tables'.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No statistically significant difference was observed in either absolute or relative thyroid weight between the control and any test substance treatment group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic abnormalities were observed in any dam.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were noted in the thyroid (including parathyroid).
In the surviving animals, ectopic thymic tissue and/or ultimobrancial remnant in the
thyroid were observed in each test substance treatment group; however, these were not judged to be treatment related because they are noted occasionally in normal rats and the same changes were also observed in the control group.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
not specified
Description (incidence and severity):
Not applicable in rats
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No statistically significant effects observed
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No statistically significant effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
No statistically significant effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
No statistically significant effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
not examined
Details on maternal toxic effects:
Death occurred in 1 dam in the 30 mg/kg group on GD 20. This animal showed no
abnormalites that directly led to the death in clinical signs, body weights, or necropsy although low food consumption was noted; thus, the direct cause of death was unknown. However, the cause of death was judged to be effects of the substance treatment by the following reasons:
(1) the surviving dams in the 30 mg/kg group showed worsening of general
conditions and lower body weight and food consumption and
(2) deaths occurred at 30 mg/kg in the end of the gestation period in reproduction/developmental toxicity screening test of the substance.

In the surviving dams, effects of the substance treatment were observed in the 30 mg/kg group as follows. In the clinical signs, decrease in locomotor activity was observed in 1 dam from GDs 19 to 20 and soiled fur (chest or abdomen) in 2 dams on GD 20. Decreased body weight and food consumption were observed in the late phase of the gestation period (GD 15 or later). No effects of the substance treatment were observed in necropsy including gravid uterus weights,organ weights, or histopathological examination (thyroid gland), or plasma hormone concentration (total T3, total T4, or TSH) at any dose level.
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
mortality
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No statistically significant effects observed
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No statistically significant effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No statistically significant effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No statistically significant effects observed
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
No statistically significant difference was observed in AGD or AGI between the control and any test substance treatment group.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In the 30 mg/kg group, anal atresia and acaudate were observed in 1 fetus (No. 566); however, these were not judged to be treatment related because these were spontaneous changes, which are occasionally seen in normal animals.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal anomalies:
No anomalies were observed in the 30 mg/kg group.

In the control group, skeletal anomalies were observed in 2 fetuses (1.4%).
With regard to individual types of skeletal anomalies, thoracic hemivertebra was observed in 1 fetus (0.7%) and short 13th rib in 1 fetus (0.7%).

Skeletal variations
No treatment-related changes were observed in the 30 mg/kg group.
Skeletal variations were observed in 23 fetuses (15.1%) and 22 fetuses (14.6%) in the control and 30 mg/kg groups, respectively. With regard to individual types of skeletal variations, the following findings were observed in the control and 30 mg/kg groups: full supernumerary rib in 3 fetuses (1.9%) and 4 fetuses (2.7%), short supernumerary rib in 13 fetuses (8.7%) and 16 fetuses (10.6%), asymmetry of the sternebra in 2 fetuses (1.2%) and 3 fetuses (2.0%), and bipartite ossification of sternebra in 2 fetuses (1.1%) and 2 fetuses (1.3%), respectively. However, no statistically significant differences were observed in their incidences between the control and 30 mg/kg groups. As the findings observed only
in the control group, bipartite ossification thoracic centrum was observed in 1 fetus (0.6%), cervical rib in 3 fetuses (2.1%), and 5 lumbar vertebrae in 1 fetus (0.7%).

Ossification progress
No statistically significant differences were observed in the number of sacrocaudal vertebral bodies or sternebrae between the control and 30 mg/kg groups.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related change were observed in the 30 mg/kg group.

Visceral anomalies were observed in 8 fetuses (5.6%) and 1 fetus (0.8%) in the control and 30 mg/kg groups, respectively. With regard to individual types of visceral anomalies, thymic remnant in the neck was observed in 2 fetuses (1.6%) and 1 fetus (0.8%) in the control and 30 mg/kg groups, respectively. As the findings observed only in the control group, dilated lateral ventricle was observed in 1 fetus (1.0%), dilated renal pelvis in 1 fetus (0.6%), and dilated ureter in 6 fetuses (3.9%).
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
No effects of the test substance were observed in examination of uterine contents (number of corpora lutea, implantations, live fetuses, or resorption, pre- implantation loss, or postimplantation loss) or examination of fetuses (sex ratio, fetal weights, AGD, or visceral, skeletal, external, or placental development).
Therefore, the substance is considered to have had no embryo-fetal lethality, growth retardation, or structural abnormalities in rats in this study.
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
no

Summary or hormone analysis measurements:




































Group



TSH (ng/mL)



Total T4 (ng/mL)



Total T3 (ng/mL)



Control



6.04 ± 2.41



15.9 ± 3.3



0.60 ± 0.11



Low dose



5.71 ± 1.93



16.2 ± 3.7



0.62 ± 0.10



Middle dose



6.32 ± 2.28



16.8 ± 3.2



0.58 ± 0.08



High dose



6.52 ± 2.17



16.3 ± 1.8



0.63 ± 0.08


Conclusions:
In conclusion, the NOAEL of the test substance was considered to be 10 mg/kg for general toxicity and reproductive function in dams, and 30 mg/kg for embryonic/fetal development under the conditions of this study.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
All toxicological studies on the test substance have been conducted in accordance to OECD (or equivalent) guideline and GLP-compliant. Therefore the quality of the database is regarded as high quality.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

The predominant effect possibly indicating reproductive toxicity was the total litter loss observed in two females in the high dose group (30 mg/kg/day). This led towards a trend for low values in gestation index, delivery index, number of live new-borns, birth index and a high value in the still-born index at this dose. However, significant mortality at this dose in the dams was noted (greater than 10%).


 


Based on the significant and treatment related effects observed in the parental females including bodyweight changes, stomach findings with relevant histopathological correlation indicative of irritation and mortality, it is considered that the total litter loss observed was related to maternal toxicity of the dams rather than inherent reproductive toxicity.


Specifically it was noted that the two dams with total litter loss showed similar gross and histopathological findings to other females that were found dead on study.


 


The CLP Regulation mentions that “maternal mortality greater than 10% is considered excessive and the data for that dose level shall not normally be considered for further evaluation”. The female mortality at 30 mg/kg/day was 25% (3/12 animals died during the study).


 


The NOAEL for parental animals on this study was deemed to be the intermediate dose (10 mg/kg/day) and on a corresponding 28 day repeated dose study the NOAEL for females was determined to be 8 mg/kg/day, with evidence of systemic toxicity observed at higher doses. Based on the effects observed these are considered to be as a result of systemic toxicity rather than solely local irritation effects in the stomach (although this has also been observed). 


 


In terms of developmental toxicity, decreased bodyweight of live new-borns was observed at the high dose (30 mg/kg/day) within the screening study, however, the substance is considered to have had no embryo-fetal lethality, growth retardation, or structural abnormalities in rats in pre-natal developmental toxicitystudy. Based on the above considerations, these findings in the screening test are also understood to be as a result of maternal toxicity rather than an inherent reproductive effect.


 


Based on the findings of in the reproductive screening test and pre-natal developmental toxicity study, this substance is not classified as a reproductive toxicant under the CLP Regulation EC No. 1272/2008, as amended).

Additional information