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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The age of the animals was not reported; no data on individual animals was given; the body weight of females was not recorded during the mating period; the time of onset, duration, number affected and severity of the clinical signs was not reported; the number of implantation sites was not recorded.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted Jul 1995
Deviations:
yes
Remarks:
animal age not reported; no data on individual animals given; time of onset, duration, number affected and severity of the clinical signs not reported; number implantation sites not given
Qualifier:
according to
Guideline:
other: The Guidelines for the Testing of Chemicals (State Environmental Protection Administration of China) 2004.5
Deviations:
not specified
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Vital River Laboratory Animal Technology Co., Ltd., Beijing, China
- Weight at study initiation: (P) Males: 225 - 305 g; Females: 205 -267 g
- Fasting period before study: no
- Housing: the animals were housed individually, except during the mating period
- Diet: reproductive diet (Certificate No. SCXK(JING) 2009-0008, HFK BIOSCIENCE Co., Ltd., Tianjin, China), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared at least once every 7 days. Each aliquot was stirred prior to dosing.

VEHICLE
- Concentration in vehicle: 50, 100 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/ kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility for a maximum of 5 days
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
(P) males: 42 days (from 14 days prior to mating, during mating until Day 43)
(P) females: up to 54 days (from 14 days prior to mating, up to 14 days during mating, up to 22 days during gestation, 4 days during lactation; for non-pregnant females, until 25 days after vaginal plug was observed)
Frequency of treatment:
daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were based on the results of the acute oral toxicity study (study report not available), in which the LD50 was > 5000 mg/kg bw.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: females: week 1 and 2 (prior to mating), gestation day 0, 7 and 14, and lactation day 0 and 4; males: week 1, 2 (prior to mating), 3, 4 and 5

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, food consumption was measured in females during week 1 and 2 (prior to mating), and gestation week 1 and 2; and in males during the whole treatment period (week 1 - 6).
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on Day 43
- Maternal animals: All surviving animals on lactation day 4; non-pregnant females were sacrificed on the same day

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations for abnormalities

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs/tissues were prepared for microscopic examination and weighed, respectively: testes, epididymes, ovaries and uterus. Any organs with lesions were prepared and subjected to a histopathological examination.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age
- These animals were subjected to postmortem examinations (macroscopic examination) as follows: all pups were examined for external and visceral abnormalities

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations for abnormalities

HISTOPATHOLOGY / ORGAN WEIGTHS
No histopathological examinations were performed.
Statistics:
Continuous data, including body weight, body weight gain, feed consumption, organ weight, pup body weight, gestation length and mean live litter size were analysed using a one-way analysis of variance (ANOVA). If significance (p < 0.05) was noted, group by group comparisons were performed using Dunnet's test. Count data were analysed using chi-square test for copulation and fertility indices, pup sex ratio, number of live and dead pups per group on lactation day 0, and pup survival after lactation day 0. All anaylses used two-tailed tests for a minimum significance level of 5% comparing the control to the treated groups. A p value of less than 0.5% was judged a significant difference.
Reproductive indices:
Mating index (%): (numbers of pairs with successful mating/ number of pairs) x 100
Fertility index (%): (Number of pregnant females/ number of pairs with successful mating) x 100
Offspring viability indices:
Live birth index (%): (Number of live pups born/ number of pups born) x 100
Viability index (%): (Number of live pups on lacation day 4/ number of live pups born) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Decreased activity, disorientation and unkempt fur was observed in most males of the high-dose group. These transient effects are not considered to be toxicologically relevant.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight and body weight gain was comparable between the treatment and control groups in males and females. The body weight increase in females of the high-dose group observed on Day 14 of gestation only, is considered to be an incidental effect.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption was significantly reduced in males of the low- and mid-dose group in week 6, and in males of the high-dose group in week 4, 5 and 6 (see Table 1 and 2 under ' Any other information on results incl. tables'). The reduced food intake did not affect the body weight, therefore, the reduced food consumption is not considered to be toxicologically relevant. The test substance intake was adjusted weekly based on the body weight and was considered by the study report authors to be close to the reported dose levels.
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The testis and epididymes weights were comparable between the treatment and control groups in males.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant differences in fertility index and dams with live young born between the control and treatment groups. The mating index was slightly, but not significantly reduced in the 500 and 1000 mg/kg bw/day groups (see Table 3 under ' Any other information on results incl. tables'). Two of nine dams in the high-dose group cannibalised their entire litter post-partum, with the study authors indicating the reason was 'abnormal maternal behaviour'. This information was not included in the English translation of the study report (only in the Chinese version) and no additional information was available. Due to the limited information given in the study report and the lack of individual animal data the possibility that this effect is treatment-related cannot be excluded. However, no toxicological relevance was evident in the general health and reproductive health of the dams and cannibalisation is known to occur in rats.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No toxicologically relevant effects were observed up to and including the highest dose level
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (sperm measures)
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The survival rate (as viability index) of pups in the high-dose group was significantly reduced, compared with the control group (see Table 3 under 'Any other information on results incl. tables'). Two of nine dams cannibalised their entire litter post-partum, which is likely to have caused the reduction in viability.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The body weight of pups in the high-dose group was significantly reduced on lactation day 0, compared with the control group. The difference was approximately 5%; indicating that the variation in body weight is incidental and not treatment-related. Furthermore, the mean weight for the control and treatment groups fell within the historical data range given for rat pups on lactation day 0 (Lang, P.L. Historical control data for development and reproductive toxicity studies using the Crl:CD BR rat. Charles River Laboratories, September 1993. www.criver.com) No significant difference was noted between the control and treatment groups on lactation day 4.
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:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The tail length of the pups in the high-dose group was significantly shorter than that of the control group. However, this is not a standard parameter and is unlikely to affect the survival and development of the pups. Therefore it is not considered to be a toxicologically relevant effect. A significant increase in pup length in the mid-dose group is considered to be incidental.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
development
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related adverse effects were observed up to and including the highest dose level

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Table 1: body weight in males

Body weight, g (mean ± SD)

 

Dose group (mg/kg bw/day)

 

Control (vehicle)

250

500

1000

Day 0

269.3 ± 17.0

268.5 ± 18.3

267.3 ± 19.4

271.7 ± 13.2

Week 1

318.1 ± 17.5

323.2 ± 14.6

320.5 ± 13.7

319.7 ± 16.2

Week 2

357.3 ± 23.5

366.0 ± 14.6

366.9 ± 17.2

357.9 ± 16.4

Week 3

416.9 ± 31.7

423.6 ± 21.6

426.3 ± 26.0

411.7 ± 21.0

Week 4

437.0 ± 33.6

443.4 ± 25.4

445.5 ± 26.5

429.3 ± 22.2

Week 5

460.7 ± 37.1

471.9 ± 28.9

474.6 ± 28.8

454.3 ± 23.5

Week 6

487.7 ± 43.0

492.5 ± 31.5

493.6 ± 32.1

475.4 ± 24.9

Table 2: food consumption in males

Food consumption, g (mean ± SD)

 

Dose group (mg/kg bw/day)

 

Control (vehicle)

250

500

1000

Week 1

176.6 ± 11.8

180.9 ± 6.5

180.9 ± 14.0

174.8 ± 9.1

Week 2

189.3 ± 20.9

187.7 ± 11.4

188.5 ± 8.2

179.7 ± 11.1

Week 4

195.4 ± 17.9

193.7 ± 20.9

188.1 ± 14.4

177 ± 14.6*

Week 5

183.6 ± 19.3

179.3 ± 20.4

174.9 ± 11.8

166 ± 17.2*

Week 6

200.0 ± 20.3

180.7 ± 18.0*

181.5 ± 11.0*

173.2 ± 15.2*

* p < 0.05

 

Table 3: reproductive performance and development data

 

Dose group (mg/kg bw/day)

 

Control (vehicle)

250

500

1000

Females showing evidence of mating (N)

15

14

10

12

Mating index (%)

100

93

67

80

Females achieving pregnancy (N)

11

11

10

9

Fertility index (%)

73

79

100

75

Dams with live young born (N)

11

11

10

9

Pups born (N)

159

154

144

137

Live pups born (N)

159

151

142

135

Live birth index (%)

100

98

99

99

Live pups on day 4 (N)

156

148

137

117

Viability index (%)

98

98

96

87*

Sex ratio (male/female)

84/74

74/77

69/73

71/64

Pup weight on day 0 (g)

6.51 ± 0.63

6.54 ± 0.77

6.67 ± 0.68

6.21 ± 0.85*

Pup weight on day 4 (g)

11.14 ± 1.80

11.43 ± 2.03

11.13 ± 1.43

11.04 ± 1.05

Pup length on day 0 (cm)

4.54 ± 0.23

4.61 ± 0.32

4.63 ± 0.26*

4.50 ± 0.30

Pup tail length on day 0 (cm)

1.74 ± 0.16

1.69 ± 0.21

1.77 ± 0.20

1.68 ± 0.16*

* p < 0.05

 

Applicant's summary and conclusion