Registration Dossier

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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

- Toxicity to reproduction, oral: NOAEL for systemic toxicity: 100 mg/kg bw/day, NOAEL for reproductive toxicity > 1000 mg/kg bw/day, male/female, rat, OECD 421, Penn 2015

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 May 2014 to 14 Oct 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Adopted 22nd January 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: (P) 8 to 9 weeks
- Weight at study initiation: (P) Males: 269 to 325 g; Females: 175 to 214 g
- Fasting period before study: No
- Housing: Animals were housed in groups of five, by sex, until pairing and for males post-pairing. For pairing, one male and one female from the same group were housed together in grid-floor cages suspended over paper-lined trays. On confirmation of mating, the males were returned to the group cages and the mated females were housed individually and subsequently with their litter, in solid floor cages with bedding material provided.
- Diet: pelleted rodent diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 22
- Humidity (%): 45 to 63
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
14 May 2014 to 14 Oct 2014
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% (w/v)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated at approximately weekly intervals (within the period of known stability), for each group separately, as a suspension in 1 % (w/v) aqueous carboxymethylcellulose. A weighed quantity of test item was added to a beaker and vehicle was gradually added whilst stirring. The suspension was removed from the stirrer and made up to final volume/weight with more vehicle and mixed with a laboratory homogeniser. For the formulations prepared for use on the first dosing occasion only, solid black flecks were visible after homogenisation and therefore the formulations were placed in an ultrasonic bath for up to 114 minutes, to ensure complete dispersion and homogeneity. After preparation, all formulations were dispensed into daily aliquots which were stored refrigerated. Formulations were stirred from at least 15 minutes before the start of dosing until completion of dosing to ensure thorough re-suspension and homogeneity.

VEHICLE
- Concentration in vehicle: 0 (control), 10, 30 and 100 mg/mL, corresponds to 0, 100, 300 and 1000 mg/kg bw/day test substance in dose formulation
- Amount of vehicle: 10 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the test substance in formulations used on the first and last days of dosing have been determined using a validated method of analysis. The concentrations and homogeneity of the test substance in the formulations used on the first dosing occasion were determined from the analysis of triplicate samples from the top, middle and bottom of each formulation. The achieved concentrations of the test substance in the formulations used on the last day of dosing were determined from the analysis of triplicate samples from the middle of each formulation.
Samples from vehicle used to dose control animals on these occasions were analysed to confirm the absence of test substance. The formulations were considered to have been accurately prepared and homogeneous (where assessed) if the measured concentrations of the test substance were within ± 10% of their nominal values with coefficients of variation no greater than 5%.
Duration of treatment / exposure:
Males were dosed once daily for 14 days before and during pairing and until the day before necropsy.
Females were dosed once daily for 14 days before pairing, during pairing and until Day 3 of lactation. Animals that were in midparturition at the time of dosing were not dosed on that day.
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected by the Sponsor after examining existing toxicity data from a 28 day toxicity study.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: daily for clinical signs of toxicity or changes in behaviour and appearance and each animal. Furthermore, twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Once each week

BODY WEIGHT:
- Time schedule for examinations: Male body weights were recorded on the first day of dosing and at weekly intervals throughout the study. Female body weights were recorded on the first day of dosing and then at weekly intervals until the day of mating. Females were also weighed on Days 0, 7, 14 and 20 of gestation and on Days 0 (where required for dose volume calculations), 1 and 4 of lactation.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

OTHER:
The females were allowed to rear their offspring to Day 4 of lactation. Abnormalities of nesting or nursing behaviour were recorded.
Oestrous cyclicity (parental animals):
During the pairing period the estrous cycle was determined by the type of cells present in the vaginal smears taken.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed after the females had littered and been necropsied
- Maternal animals: All surviving animals were sacrificed on Day 4 of lactation

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the thoracic, and abdominal viscera. For females, the uterus was examined and the number of implantation scars recorded on Day 4 of lactation.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 in ‘Any other information on materials and methods incl. tables’ were prepared for microscopic examination and weighed, respectively.
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:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the thoracic and abdominal viscera.
Statistics:
Data were processed to give group mean values and standard deviations, where appropriate. Where the data allowed, the following methods were used for statistical analysis comparing Groups 2, 3 and 4 against Group 1.
General Approach: All statistical tests were two-sided with minimum significance levels of 5% and 1%. Non-parametric statistics were not routinely conducted. When used, Dunnett’s test was conducted regardless of the outcome of the analysis of variance (ANOVA) or analysis of covariance (ANCOVA). After examining for any outliers, if the variances were clearly heterogeneous, transformations were used in an attempt to stabilise the variances.
For Quantitative Data: Body weight, food intake, cumulative body weight gain from the start of dosing (throughout gestation and lactation), pup body weights, cumulative pup body weight gain, litter size, gestation length and total litter weight were analysed using ANOVA. Parental organ weights were analysed using ANOVA for the absolute weights and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate.
For Percentages: Pup survival, pup sex ratios and litter based mean percentages were analysed using a parametric ANOVA, following a double arcsine transformation.
Dunnett’s test: For all of the parameters evaluated initially by ANOVA or ANCOVA, Dunnett’s test was used to compare the control and treated groups, based on the error mean square in the ANOVA or ANCOVA. The Dunnett’s test was performed for all continuous data parameters, regardless of whether the initial ANOVA or ANCOVA was statistically significant, and statistical flags are presented in the tables of results in the final report.
Reproductive indices:
See Table 2 in 'Any other information on materials and methods incl. tables'.
Offspring viability indices:
See Table 2 in 'Any other information on materials and methods incl. tables'.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related clinical observations.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female animal, administered 1000 mg/kg/day, was killed on Day 0 of lactation due to poor clinical condition within 102 minutes of dosing. Littering had been completed successfully and 13 live pups born. However, observations post dosing included decreased activity, prostrate posture and slow breathing indicative of a possible mis-dose although there were no macroscopic necropsy findings to confirm this. This death was an isolated incidence and, in the absence of other deaths or clinical signs of toxicity at 1000 mg/kg/day in this study or in a 28-day toxicity study, was considered to be incidental and not related to treatment with the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Throughout the dosing period, males given 1000 mg/kg/day had reduced body weight gains (p<0.01) compared with Controls (-29 %). Males given 300 mg/kg/day had lower body weight gains (-9 %) throughout the dosing period compared with Controls, although the difference was not statistically significant. There was no effect on body weight or body weight gains for males at 100 mg/kg/day or for females during pre-pairing, gestation or lactation, at any dose level.
See Tables 1 to 4 in ‘Any other information on results incl. tables’.
Food consumption and compound intake (if feeding study):
no effects observed
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
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related microscopic changes were found in the liver of males and females and in the thyroid of males. These changes are described below. The spectrum of all other microscopic findings was consistent with changes encountered in rats of this age kept under laboratory conditions and none was considered to be treatment-related
Liver: Hepatocellular hypertrophy (minimal) was present in all males and 2/10 females given 1000 mg/kg/day and in 2/10 males given 300 mg/kg/day. The incidence of the treatment related change was dose-related in males and accounted for the observed dose-related increases in liver weight in both sexes.
Thyroids: There was an increased incidence of thyroid follicular hypertrophy (minimal) in males given 300 mg/kg/day or 1000 mg/kg/day when compared with males from the Control group. This increased incidence was considered to be treatment-related. Although the incidence of follicular cell hypertrophy in males given 100 mg/kg/day was greater than that found in the concurrent Controls, the finding was considered to be within the spectrum of change encountered in male rats of this strain kept under laboratory conditions and therefore incidental to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Fertility and mating performance were unaffected by the test substance, all animals mated within four days of pairing, with no effects on the pregnancy parameters. There was no effect of treatment on the mean duration of gestation, with all pregnant females giving birth to live litters.
See Tables 6 and 7 in ‘Any other information on results incl. tables’.
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
See Table 5 in ‘Any other information on results incl. tables’.
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
Description (incidence and severity):
There were no treatment-related macroscopic findings.
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Formulation analysis

Test item formulations from the first preparation occasion and those used on the last day of dosing were considered to have been accurately prepared and homogeneous (where assessed) since the measured concentrations of the test substance were within 5 % of their nominal values with coefficients of variation no greater than 1.3 % which fulfilled the acceptance criteria (± 10 % and ≤ 5 % for accuracy and homogeneity, respectively).

No test substance was detected in vehicle used to dose Control animals.

Table 1. Body Weights (g) - P Generation Males - Group Mean Values

Sex: Male

Body Weight

(Week Number)

Group

0#

1#

2#

3#

4#

5#

6#

Group: 1 Control

0 mg/kg/day

Mean

294.0

320.0

340.7

353.6

365.0

380.0

392.3

SD

14.0

16.1

17.9

16.6

18.9

19.6

16.6

N

10

10

10

10

10

10

10

Group: 2

100 mg/kg/day

Mean

295.6

324.9

348.7

354.8

377.8

390.9

402.5

SD

17.8

20.6

21.7

16.2

22.8

21.0

22.7

N

10

10

10

10

10

10

10

Group: 3

300 mg/kg/day

Mean

288.4

308.6

328.6

341.3

352.6

368.4

377.9

SD

18.5

21.6

25.9

26.3

24.6

31.1

28.6

N

10

10

10

10

10

10

10

Group: 4

1000 mg/kg/day

Mean

291.5

301.1

321.4

335.1

345.8

352.0 d1

361.5 d1

SD

15.1

23.5

23.8

24.1

25.4

25.4

26.0

N

10

10

10

10

10

10

10

# [Statistically Analysed]

1 [d - Test: Dunnett 2 Sided p < 0.05]

 

Table 2. Body Weights (g) - P Generation Females - Pre-Pairing - Group Mean Values

 

Sex: Female

Body Weight

(Week Number)

Group

0#

1#

2#

Group: 1 Control

0 mg/kg/day

Mean

189.4

198.2

209.0

SD

7.1

7.1

8.5

N

10

10

10

Group: 2

100 mg/kg/day

Mean

195.7

203.3

213.6

SD

10.7

11.1

11.2

N

10

10

10

Group: 3

300 mg/kg/day

Mean

193.9

201.3

216.7

SD

11.7

12.6

11.0

N

10

10

10

Group: 4

1000 mg/kg/day

Mean

188.9

202.8

209.3

SD

5.8

9.2

9.0

N

9

9

9

# [Statistically Analysed]

 

Table 3. Body Weights (g) - P Generation Females - Gestation - Group Mean Values

 

Sex: Female

Body Weight

(Day of Gestation)

Group

0#

7#

14#

20#

Group: 1 Control

0 mg/kg/day

Mean

209.1

238.0

267.1

329.0

SD

10.2

13.2

18.0

22.7

N

10

10

10

10

Group: 2

100 mg/kg/day

Mean

217.1

241.1

270.3

329.2

SD

11.1

11.6

12.8

18.4

N

10

10

10

10

Group: 3

300 mg/kg/day

Mean

217.6

243.8

272.3

327.1

SD

9.2

11.2

13.2

14.4

N

10

10

10

10

Group: 4

1000 mg/kg/day

Mean

212.4

239.9

267.8

323.6

SD

8.6

9.1

11.2

15.6

N

9

9

9

9

# [Statistically Analysed]

 

Table 4. Body Weights (g) - P Generation Females - Lactation - Group Mean Values

 

Sex: Female

Body Weight

(Day of Lactation)

Group

1#

4#

Group: 1 Control

0 mg/kg/day

Mean

250.0

262.8

SD

17.0

17.5

N

10

10

Group: 2

100 mg/kg/day

Mean

253.5

270.3

SD

14.1

15.9

N

10

10

Group: 3

300 mg/kg/day

Mean

253.6

270.2

SD

15.1

14.9

N

10

10

Group: 4

1000 mg/kg/day

Mean

249.2

265.3

SD

13.6

12.3

N

9

9

# [Statistically Analysed]

 

Table 5. Body Weights (g) - F1 Generation Pups

 

Sex: Female

Mean Pup

BWt M+F (Day of Lactation)

Weight Gain

(Day of Lactation)

Group

1#

4#

1 to 4#

Group: 1 Control

0 mg/kg/day

Mean

6.59

9.89

3.30

SD

0.95

1.68

0.82

N

10

10

10

Group: 2

100 mg/kg/day

Mean

6.61

10.14

3.53

SD

0.33

0.57

0.32

N

10

10

10

Group: 3

300 mg/kg/day

Mean

6.24

9.35

3.10

SD

0.79

1.18

0.50

N

10

10

10

Group: 4

1000 mg/kg/day

Mean

6.76

9.94

3.18

SD

0.35

0.78

0.61

N

9

9

9

Data are recorded and reported against dams, day of lactation is equivalent to day of age

# [Statistically Analysed]

 

Table 6. Fertility and Mating Data - P Generation - Group Mean Values

Sex: Both

Group: 1 Control

0 mg/kg/day

Group: 2

100 mg/kg/day

Group: 3

300 mg/kg/day

Group: 4

1000 mg/kg/day

Number of Females Paired

N+ve

10

10

10

10

Number of Females Mated

N+ve

10

10

10

10

Number of Fertile Females

N+ve

10

10

10

10

Copulation Index Female %#

Mean

100.0

100.0

100.0

100.0

Female Fertility Index %#

Mean

100.0

100.0

100.0

100.0

Number of Males Paired

N+ve

10

10

10

10

Number of Males Mated

N+ve

10

10

10

10

Number of Fertile Males

N+ve

10

10

10

10

Copulation Index Male %#

Mean

100.0

100.0

100.0

100.0

Male Fertility Index %#

Mean

100.0

100.0

100.0

100.0

# [Statistically Analysed]

 

Table 7. Pregnancy and Litter Data - P Generation Females and Litters - Group Mean Values

Sex: Female

Group: 1 Control

0 mg/kg/day

Group: 2

100 mg/kg/day

Group: 3

300 mg/kg/day

Group: 4

1000 mg/kg/day

Gestation Length (day)#

Mean

22.30

22.05

22.15

22.17

Gestation Index #

%

100.0

100.0

100.0

100.0

Total No of Implantation Scars

Mean

13.7

13.3

13.4

13.0

Total Pups Born#

Mean

13.0

12.3

12.6

11.8

Post Implantation Loss %#

Mean

5.3

7.4

7.0

8.9

Live Pups on Day 0

Mean

13.0

12.3

12.6

11.8

Live Pups on Day 1

Mean

12.9

12.2

12.4

11.8

Live Pups Day 4 (Pre Cull)

Mean

12.9

12.2

12.4

11.7

Live Birth Index (%)#

Mean

100.00

100.00

100.00

100.00

Viability Index 1 (%)#

Mean

99.23

99.00

98.50

99.15

Cumulative Survival Index % #

Mean

99.23

99.00

98.50

99.15

% Males#

Mean

56.57

42.85

54.10

51.24

# [Statistically Analysed]

 

Table 8. Litter Necropsy Findings - F1 Generation - Summary

 

Sex : Females

Group: 1 Control

0 mg/kg/day

Group: 2

100 mg/kg/day

Group: 3

300 mg/kg/day

Group: 4

1000 mg/kg/day

Total number of litters

10

10

10

10

 

 

 

 

 

Number of pups

 

 

 

 

 

 

 

 

 

Born

130

123

126

119

Found dead/killed prematurely

0

0

1

13

Missing (presumed cannibalised)

1

1

1

1

Abnormalities

0

0

0

1

Left kidney pelvic dilation severe left uterer distended severe

0

0

0

1

 

Table 9. Organ Weights (g) - P Generation Males - Group Mean Values

Sex: Male

Dead

Body Weight

Absolute

Liver Weight

Adjusted

Liver Weight

Liver %

Body Weight

Absolute Testes

Weight

Adjusted

Testes wt

Testes %

Body Weight

Absolute Epididymides

Weight

Adjusted

Epididymides wt

Epididymides

% Body Weight

Absolute Thyroids

Weight

Adjusted

Thyroids wt

Thyroids %

Body Weight

Group

#

#

#

 

#

#

 

#

#

 

#

#

 

Group: 1 Control

0 mg/kg/day

Mean

401.79

13.744

13.33

3.42

3.722

3.680

0.928

1.352

1.334

0.337

0.0217

0.0210

0.0054

SD

18.57

0.881

 

0.20

0.428

 

0.111

0.146

 

0.037

0.0037

 

0.0010

N

10

10

10

10

10

10

10

10

10

10

10

10

10

Group: 2

100 mg/kg/day

Mean

413.45

14.572

13.66

3.52

3.527

3.436

0.854

1.304

1.265

0.315

0.0224

0.0210

0.0054

SD

24.52

1.010

 

0.14

0.299

 

0.071

0.121

 

0.022

0.0032

 

0.0007

N

10

10

10

10

10

10

10

10

10

10

10

10

10

Group: 3

300 mg/kg/day

Mean

385.64

14.826

15.09

dd2

3.83

3.470

3.497

0.903

1.272

1.283

0.332

0.0222

0.0220

0.0058

SD

32.22

2.185

 

0.28

0.287

 

0.079

0.129

 

0.040

0.0033

 

0.0008

N

10

10

10

10

10

10

10

10

10

10

10

10

10

Group: 4

1000 mg/kg/day

Mean

367.01

d1

15.431

d1

16.49

dd2

4.21

3.555

3.661

0.971

1.253

1.298

0.342

0.0222

0.0230

0.0060

SD

28.81

1.129

 

0.20

0.299

 

0.078

0.151

 

0.035

0.0041

 

0.0009

N

10

10

10

10

10

10

10

10

10

10

10

10

10

# [Statistically Analysed]

1 [d - Test: Dunnett 2 Sided p < 0.05] 2 [dd - Test: Dunnett 2 Sided p < 0.01]

 

Table 10. Organ Weights (g) - P Generation Females - Group Mean Values

Sex: Female

Dead

Body Weight

Absolute

Liver Weight

Adjusted

Liver Weight

Liver %

Body Weight

Absolute

Thyroids wt

Adjusted

Thyroids wt

Thyroids %

Body Weight

Group

#

#

#

 

#

#

 

Group: 1 Control

0 mg/kg/day

Mean

261.23

11.405

11.60

4.37

0.0157

0.0159

0.0060

SD

18.65

1.266

 

0.36

0.0033

 

0.0011

N

10

10

10

10

10

10

10

Group: 2

100 mg/kg/day

Mean

267.91

11.783

11.64

4.39

0.0187

0.0186

0.0070

SD

15.92

1.108

 

0.25

0.0031

 

0.0010

N

10

10

10

10

10

10

10

Group: 3

300 mg/kg/day

Mean

268.53

12.153

11.98

4.52

0.0194

d2

0.0192

0.0072

SD

11.51

1.113

 

0.31

0.0033

 

0.0013

N

10

10

10

10

10

10

10

Group: 4

1000 mg/kg/day

Mean

262.23

12.979

dd1

13.12

dd1

4.95

0.0181

0.0183

0.0069

SD

12.13

0.780

 

0.25

0.0032

 

0.0014

N

9

9

9

9

9

9

9

# [Statistically Analysed]

1 [dd - Test: Dunnett 2 Sided p < 0.01] 2 [d - Test: Dunnett 2 Sided p < 0.05]

 

Conclusions:
In this GLP compliant OECD 421 study, a reproduction/ developmental toxicity screening was performed on male and female Crl:WI(Han) mice. The following NOAELs were found:
- Systemic toxicity: 100 mg/kg bw/day based on reduced body weight, increased liver weights and minimal hepatocellular hypertrophy in males.
- Reproductive toxicity: > 1000 mg/kg bw/day
Executive summary:

In this GLP compliant OECD 421 study, a reproduction/ developmental toxicity screening was performed. Forty male and 40 female rats of the Crl:WI(Han) strain were allocated to the study. Groups of 10 males and 10 females were dosed with 0 (vehicle), 100, 300 or 1000 mg/kg/day test substance, once daily, by oral gavage at a dose volume of 10 mL/kg body weight using 1% aqueous carboxymethylcellulose as vehicle. Males were dosed for 14 days before and during pairing and until the day before necropsy (47 days) and the females were dosed for 14 days before pairing, during pairing and then until Day 3 of lactation (approximately 42 days). All animals were examined for effects on general condition, body weight and food intake. During the pairing period vaginal smears were taken daily until sperm were found in the smear. The females were allowed to litter and rear their offspring to Day 4 of age. A macroscopic necropsy was performed on all parental animals and F1 generation pups. For the parental males and females a selection of organs were weighed, fixed and examined microscopically.

There were no deaths considered to be related to treatment with the test item and no test-item related clinical signs. Throughout the dosing period, males given 300 mg/kg/day or 1000 mg/kg/day showed reduced body weight gain, compared with Controls. There was no effect on body weight or body weight gain for females at any dose level or for males given 100 mg/kg/day. There was no effect on food intake for males or females at any dose level. Fertility and mating performance were unaffected by treatment with the test substance and there were no effects on pregnancy parameters. Males given 300 mg/kg/day and both sexes given 1000 mg/kg/day showed increased liver weights adjusted for body weight. Minimal hepatocellular hypertrophy was noted in males given 300 mg/kg/day and in both sexes given 1000 mg/kg/day. Minimal thyroid follicular hypertrophy was also seen in males given 300 or 1000 mg/kg/day. There was no effect of maternal treatment with the test substance on pup body weights to Day 4 of age. Pup survival was similar across all groups including the Controls.

In conclusion, the test substance administered once daily by oral gavage to male and female rats at doses of 0, 100, 300 or 1000 mg/kg/day was well tolerated at all dose levels. The No Observed Adverse Effect Level (NOAEL) for male and female reproductive performance was 1000 mg/kg/day. The NOAEL for parental toxicity was 100 mg/kg/day. Reduced body weight and thyroid follicular hypertrophy were observed at the higher dose levels in males only. Increased liver weight in association with minimal hepatocellular hypertrophy was observed at 300 (males only) and 1000 mg/kg/day, in both sexes with males being more severely affected than females.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP compliant OECD 421 study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to reproduction

In this GLP compliant OECD 421 study, a reproduction/ developmental toxicity screening was performed. Forty male and 40 female rats of the Crl:WI(Han) strain were allocated to the study. Groups of 10 males and 10 females were dosed with 0 (vehicle), 100, 300 or 1000 mg/kg/day test substance, once daily, by oral gavage at a dose volume of 10 mL/kg body weight using 1% aqueous carboxymethylcellulose as vehicle. Males were dosed for 14 days before and during pairing and until the day before necropsy (47 days) and the females were dosed for 14 days before pairing, during pairing and then until Day 3 of lactation (approximately 42 days). All animals were examined for effects on general condition, body weight and food intake. During the pairing period vaginal smears were taken daily until sperm were found in the smear. The females were allowed to litter and rear their offspring to Day 4 of age. A macroscopic necropsy was performed on all parental animals and F1 generation pups. For the parental males and females a selection of organs were weighed, fixed and examined microscopically.

There were no deaths considered to be related to treatment with the test item and no test-item related clinical signs. Throughout the dosing period, males given 300 mg/kg/day or 1000 mg/kg/day showed reduced body weight gain, compared with Controls. There was no effect on body weight or body weight gain for females at any dose level or for males given 100 mg/kg/day. There was no effect on food intake for males or females at any dose level. Fertility and mating performance were unaffected by treatment with the test substance and there were no effects on pregnancy parameters. Males given 300 mg/kg/day and both sexes given 1000 mg/kg/day showed increased liver weights adjusted for body weight. Minimal hepatocellular hypertrophy was noted in males given 300 mg/kg/day and in both sexes given 1000 mg/kg/day. Minimal thyroid follicular hypertrophy was also seen in males given 300 or 1000 mg/kg/day. There was no effect of maternal treatment with the test substance on pup body weights to Day 4 of age. Pup survival was similar across all groups including the Controls.

In conclusion, the test substance administered once daily by oral gavage to male and female rats at doses of 0, 100, 300 or 1000 mg/kg/day was well tolerated at all dose levels. The No Observed Adverse Effect Level (NOAEL) for male and female reproductive performance was 1000 mg/kg/day. The NOAEL for parental toxicity was 100 mg/kg/day. Reduced body weight and thyroid follicular hypertrophy were observed at the higher dose levels in males only. Increased liver weight in association with minimal hepatocellular hypertrophy was observed at 300 (males only) and 1000 mg/kg/day, in both sexes with males being more severely affected than females. The NOAEL for toxicity to the offspring was 1000 mg/kg/day.

Effects on developmental toxicity

Description of key information

- Developmental toxicity, oral: NOAEL for systemic toxicity: 100 mg/kg bw/day, NOAEL for reproductive and developmental toxicity > 1000 mg/kg bw/day, male/female, rat, OECD 421, Penn 2015

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 May 2014 to 14 Oct 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for the Testing of Chemicals, No. 421
Version / remarks:
Adopted 22nd January 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3550 Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: (P) 8 to 9 weeks
- Weight at study initiation: (P) Males: 269 to 325 g; Females: 175 to 214 g
- Fasting period before study: No
- Housing: Animals were housed in groups of five, by sex, until pairing and for males post-pairing. For pairing, one male and one female from the same group were housed together in grid-floor cages suspended over paper-lined trays. On confirmation of mating, the males were returned to the group cages and the mated females were housed individually and subsequently with their litter, in solid floor cages with bedding material provided.
- Diet: pelleted rodent diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 22
- Humidity (%): 45 to 63
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
14 May 2014 to 14 Oct 2014
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% (w/v)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated at approximately weekly intervals (within the period of known stability), for each group separately, as a suspension in 1 % (w/v) aqueous carboxymethylcellulose. A weighed quantity of test item was added to a beaker and vehicle was gradually added whilst stirring. The suspension was removed from the stirrer and made up to final volume/weight with more vehicle and mixed with a laboratory homogeniser. For the formulations prepared for use on the first dosing occasion only, solid black flecks were visible after homogenisation and therefore the formulations were placed in an ultrasonic bath for up to 114 minutes, to ensure complete dispersion and homogeneity. After preparation, all formulations were dispensed into daily aliquots which were stored refrigerated. Formulations were stirred from at least 15 minutes before the start of dosing until completion of dosing to ensure thorough re-suspension and homogeneity.

VEHICLE
- Concentration in vehicle: 0 (control), 10, 30 and 100 mg/mL, corresponds to 0, 100, 300 and 1000 mg/kg bw/day test substance in dose formulation
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the test substance in formulations used on the first and last days of dosing have been determined using a validated method of analysis. The concentrations and homogeneity of the test substance in the formulations used on the first dosing occasion were determined from the analysis of triplicate samples from the top, middle and bottom of each formulation. The achieved concentrations of the test substance in the formulations used on the last day of dosing were determined from the analysis of triplicate samples from the middle of each formulation.
Samples from vehicle used to dose control animals on these occasions were analysed to confirm the absence of test substance. The formulations were considered to have been accurately prepared and homogeneous (where assessed) if the measured concentrations of the test substance were within ± 10% of their nominal values with coefficients of variation no greater than 5%.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually
Duration of treatment / exposure:
Males were dosed once daily for 14 days before and during pairing and until the day before necropsy.
Females were dosed once daily for 14 days before pairing, during pairing and until Day 3 of lactation. Animals that were in midparturition at the time of dosing were not dosed on that day.
Frequency of treatment:
Once daily
Duration of test:
Males were dosed once daily for 14 days before and during pairing and until the day before necropsy.
Females were dosed once daily for 14 days before pairing, during pairing and until Day 3 of lactation. Animals that were in midparturition at the time of dosing were not dosed on that day.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected by the Sponsor after examining existing toxicity data from a 28 day toxicity study.
Maternal examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: daily for clinical signs of toxicity or changes in behaviour and appearance and each animal. Furthermore, twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Once each week

BODY WEIGHT:
- Time schedule for examinations: Male body weights were recorded on the first day of dosing and at weekly intervals throughout the study. Female body weights were recorded on the first day of dosing and then at weekly intervals until the day of mating. Females were also weighed on Days 0, 7, 14 and 20 of gestation and on Days 0 (where required for dose volume calculations), 1 and 4 of lactation.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

POST-MORTEM EXAMINATIONS:
- Male animals: All surviving animals were sacrificed after the females had littered and been necropsied
- Maternal animals: All surviving animals were sacrificed on Day 4 of lactation
- Organs examined: Gross necropsy consisted of external and internal examinations including the thoracic, and abdominal viscera. For females, the uterus was examined and the number of implantation scars recorded on Day 4 of lactation.
- Histopathology and organ weights: The tissues indicated in Table 1 in ‘Any other information on materials and methods incl. tables’ were prepared for microscopic examination and weighed, respectively

OTHER:
The females were allowed to rear their offspring to Day 4 of lactation. Abnormalities of nesting or nursing behaviour were recorded.
Ovaries and uterine content:
The uterine content was examined after termination:
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: No
- Head examinations: No
Statistics:
Data were processed to give group mean values and standard deviations, where appropriate. Where the data allowed, the following methods were used for statistical analysis comparing Groups 2, 3 and 4 against Group 1.
General Approach: All statistical tests were two-sided with minimum significance levels of 5% and 1%. Non-parametric statistics were not routinely conducted. When used, Dunnett’s test was conducted regardless of the outcome of the analysis of variance (ANOVA) or analysis of covariance (ANCOVA). After examining for any outliers, if the variances were clearly heterogeneous, transformations were used in an attempt to stabilise the variances.
For Quantitative Data: Body weight, food intake, cumulative body weight gain from the start of dosing (throughout gestation and lactation), pup body weights, cumulative pup body weight gain, litter size, gestation length and total litter weight were analysed using ANOVA. Parental organ weights were analysed using ANOVA for the absolute weights and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate.
For Percentages: Pup survival, pup sex ratios and litter based mean percentages were analysed using a parametric ANOVA, following a double arcsine transformation.
Dunnett’s test: For all of the parameters evaluated initially by ANOVA or ANCOVA, Dunnett’s test was used to compare the control and treated groups, based on the error mean square in the ANOVA or ANCOVA. The Dunnett’s test was performed for all continuous data parameters, regardless of whether the initial ANOVA or ANCOVA was statistically significant, and statistical flags are presented in the tables of results in the final report.
Indices:
See Table 2 in 'Any other information on materials and methods incl. tables'.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related clinical observations.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female animal, administered 1000 mg/kg/day, was killed on Day 0 of lactation due to poor clinical condition within 102 minutes of dosing. Littering had been completed successfully and 13 live pups born. However, observations post dosing included decreased activity, prostrate posture and slow breathing indicative of a possible mis-dose although there were no macroscopic necropsy findings to confirm this. This death was an isolated incidence and, in the absence of other deaths or clinical signs of toxicity at 1000 mg/kg/day in this study or in a 28-day toxicity study, was considered to be incidental and not related to treatment with the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Throughout the dosing period, males given 1000 mg/kg/day had reduced body weight gains (p<0.01) compared with Controls (-29 %). Males given 300 mg/kg/day had lower body weight gains (-9 %) throughout the dosing period compared with Controls, although the difference was not statistically significant. There was no effect on body weight or body weight gains for males at 100 mg/kg/day or for females during pre-pairing, gestation or lactation, at any dose level.
See Tables 1 to 4 in ‘Any other information on results incl. tables’.
Food consumption and compound intake (if feeding study):
no effects observed
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):
There were statistically significant increases in liver weight adjusted for body weight (p<0.01) in the males administered 300 mg/kg/day (+13 %) and in males and females administered 1000 mg/kg/day (+24 % and +13 %, respectively), compared with the Controls.
There was no effect of the test substanceon testes, epididymides or thyroid weights.
See Tables 9 and 10 in ‘Any other information on results incl. tables’.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The spectrum of all macroscopic findings was consistent with changes encountered in rats of this age kept under laboratory conditions and there was no evidence of any treatment-related effects.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related microscopic changes were found in the liver of males and females and in the thyroid of males. These changes are described below. The spectrum of all other microscopic findings was consistent with changes encountered in rats of this age kept under laboratory conditions and none was considered to be treatment-related.
Liver: Hepatocellular hypertrophy (minimal) was present in all males and 2/10 females given 1000 mg/kg/day and in 2/10 males given 300 mg/kg/day. The incidence of the treatment related change was dose-related in males and accounted for the observed dose-related increases in liver weight in both sexes.
Thyroids: There was an increased incidence of thyroid follicular hypertrophy (minimal) in males given 300 mg/kg/day or 1000 mg/kg/day when compared with males from the Control group. This increased incidence was considered to be treatment-related. Although the incidence of follicular cell hypertrophy in males given 100 mg/kg/day was greater than that found in the concurrent Controls, the finding was considered to be within the spectrum of change encountered in male rats of this strain kept under laboratory conditions and therefore incidental to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no effect on the number of implantation scars or the incidence of postimplantation loss.
See Table 7 in ‘Any other information on results incl. tables’.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no total litter losses.
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
See Table 7 in ‘Any other information on results incl. tables’.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
See Table 7 in ‘Any other information on results incl. tables’.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity (males)
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity (females)
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
See Table 5 in ‘Any other information on results incl. tables’.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
See Table 8 in ‘Any other information on results incl. tables’.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Formulation analysis

Test item formulations from the first preparation occasion and those used on the last day of dosing were considered to have been accurately prepared and homogeneous (where assessed) since the measured concentrations of the test substance were within 5 % of their nominal values with coefficients of variation no greater than 1.3 % which fulfilled the acceptance criteria (± 10 % and ≤ 5 % for accuracy and homogeneity, respectively).

No test substance was detected in vehicle used to dose Control animals.

Table 1. Body Weights (g) - P Generation Males - Group Mean Values

Sex: Male

Body Weight

(Week Number)

Group

0#

1#

2#

3#

4#

5#

6#

Group: 1 Control

0 mg/kg/day

Mean

294.0

320.0

340.7

353.6

365.0

380.0

392.3

SD

14.0

16.1

17.9

16.6

18.9

19.6

16.6

N

10

10

10

10

10

10

10

Group: 2

100 mg/kg/day

Mean

295.6

324.9

348.7

354.8

377.8

390.9

402.5

SD

17.8

20.6

21.7

16.2

22.8

21.0

22.7

N

10

10

10

10

10

10

10

Group: 3

300 mg/kg/day

Mean

288.4

308.6

328.6

341.3

352.6

368.4

377.9

SD

18.5

21.6

25.9

26.3

24.6

31.1

28.6

N

10

10

10

10

10

10

10

Group: 4

1000 mg/kg/day

Mean

291.5

301.1

321.4

335.1

345.8

352.0 d1

361.5 d1

SD

15.1

23.5

23.8

24.1

25.4

25.4

26.0

N

10

10

10

10

10

10

10

# [Statistically Analysed]

1 [d - Test: Dunnett 2 Sided p < 0.05]

 

Table 2. Body Weights (g) - P Generation Females - Pre-Pairing - Group Mean Values

 

Sex: Female

Body Weight

(Week Number)

Group

0#

1#

2#

Group: 1 Control

0 mg/kg/day

Mean

189.4

198.2

209.0

SD

7.1

7.1

8.5

N

10

10

10

Group: 2

100 mg/kg/day

Mean

195.7

203.3

213.6

SD

10.7

11.1

11.2

N

10

10

10

Group: 3

300 mg/kg/day

Mean

193.9

201.3

216.7

SD

11.7

12.6

11.0

N

10

10

10

Group: 4

1000 mg/kg/day

Mean

188.9

202.8

209.3

SD

5.8

9.2

9.0

N

9

9

9

# [Statistically Analysed]

 

Table 3. Body Weights (g) - P Generation Females - Gestation - Group Mean Values

 

Sex: Female

Body Weight

(Day of Gestation)

Group

0#

7#

14#

20#

Group: 1 Control

0 mg/kg/day

Mean

209.1

238.0

267.1

329.0

SD

10.2

13.2

18.0

22.7

N

10

10

10

10

Group: 2

100 mg/kg/day

Mean

217.1

241.1

270.3

329.2

SD

11.1

11.6

12.8

18.4

N

10

10

10

10

Group: 3

300 mg/kg/day

Mean

217.6

243.8

272.3

327.1

SD

9.2

11.2

13.2

14.4

N

10

10

10

10

Group: 4

1000 mg/kg/day

Mean

212.4

239.9

267.8

323.6

SD

8.6

9.1

11.2

15.6

N

9

9

9

9

# [Statistically Analysed]

 

Table 4. Body Weights (g) - P Generation Females - Lactation - Group Mean Values

 

Sex: Female

Body Weight

(Day of Lactation)

Group

1#

4#

Group: 1 Control

0 mg/kg/day

Mean

250.0

262.8

SD

17.0

17.5

N

10

10

Group: 2

100 mg/kg/day

Mean

253.5

270.3

SD

14.1

15.9

N

10

10

Group: 3

300 mg/kg/day

Mean

253.6

270.2

SD

15.1

14.9

N

10

10

Group: 4

1000 mg/kg/day

Mean

249.2

265.3

SD

13.6

12.3

N

9

9

# [Statistically Analysed]

 

Table 5. Body Weights (g) - F1 Generation Pups

 

Sex: Female

Mean Pup

BWt M+F (Day of Lactation)

Weight Gain

(Day of Lactation)

Group

1#

4#

1 to 4#

Group: 1 Control

0 mg/kg/day

Mean

6.59

9.89

3.30

SD

0.95

1.68

0.82

N

10

10

10

Group: 2

100 mg/kg/day

Mean

6.61

10.14

3.53

SD

0.33

0.57

0.32

N

10

10

10

Group: 3

300 mg/kg/day

Mean

6.24

9.35

3.10

SD

0.79

1.18

0.50

N

10

10

10

Group: 4

1000 mg/kg/day

Mean

6.76

9.94

3.18

SD

0.35

0.78

0.61

N

9

9

9

Data are recorded and reported against dams, day of lactation is equivalent to day of age

# [Statistically Analysed]

 

Table 6. Fertility and Mating Data - P Generation - Group Mean Values

Sex: Both

Group: 1 Control

0 mg/kg/day

Group: 2

100 mg/kg/day

Group: 3

300 mg/kg/day

Group: 4

1000 mg/kg/day

Number of Females Paired

N+ve

10

10

10

10

Number of Females Mated

N+ve

10

10

10

10

Number of Fertile Females

N+ve

10

10

10

10

Copulation Index Female %#

Mean

100.0

100.0

100.0

100.0

Female Fertility Index %#

Mean

100.0

100.0

100.0

100.0

Number of Males Paired

N+ve

10

10

10

10

Number of Males Mated

N+ve

10

10

10

10

Number of Fertile Males

N+ve

10

10

10

10

Copulation Index Male %#

Mean

100.0

100.0

100.0

100.0

Male Fertility Index %#

Mean

100.0

100.0

100.0

100.0

# [Statistically Analysed]

 

Table 7. Pregnancy and Litter Data - P Generation Females and Litters - Group Mean Values

Sex: Female

Group: 1 Control

0 mg/kg/day

Group: 2

100 mg/kg/day

Group: 3

300 mg/kg/day

Group: 4

1000 mg/kg/day

Gestation Length (day)#

Mean

22.30

22.05

22.15

22.17

Gestation Index #

%

100.0

100.0

100.0

100.0

Total No of Implantation Scars

Mean

13.7

13.3

13.4

13.0

Total Pups Born#

Mean

13.0

12.3

12.6

11.8

Post Implantation Loss %#

Mean

5.3

7.4

7.0

8.9

Live Pups on Day 0

Mean

13.0

12.3

12.6

11.8

Live Pups on Day 1

Mean

12.9

12.2

12.4

11.8

Live Pups Day 4 (Pre Cull)

Mean

12.9

12.2

12.4

11.7

Live Birth Index (%)#

Mean

100.00

100.00

100.00

100.00

Viability Index 1 (%)#

Mean

99.23

99.00

98.50

99.15

Cumulative Survival Index % #

Mean

99.23

99.00

98.50

99.15

% Males#

Mean

56.57

42.85

54.10

51.24

# [Statistically Analysed]

 

Table 8. Litter Necropsy Findings - F1 Generation - Summary

 

Sex : Females

Group: 1 Control

0 mg/kg/day

Group: 2

100 mg/kg/day

Group: 3

300 mg/kg/day

Group: 4

1000 mg/kg/day

Total number of litters

10

10

10

10

 

 

 

 

 

Number of pups

 

 

 

 

 

 

 

 

 

Born

130

123

126

119

Found dead/killed prematurely

0

0

1

13

Missing (presumed cannibalised)

1

1

1

1

Abnormalities

0

0

0

1

Left kidney pelvic dilation severe left uterer distended severe

0

0

0

1

 

Table 9. Organ Weights (g) - P Generation Males - Group Mean Values

Sex: Male

Dead

Body Weight

Absolute

Liver Weight

Adjusted

Liver Weight

Liver %

Body Weight

Absolute Testes

Weight

Adjusted

Testes wt

Testes %

Body Weight

Absolute Epididymides

Weight

Adjusted

Epididymides wt

Epididymides

% Body Weight

Absolute Thyroids

Weight

Adjusted

Thyroids wt

Thyroids %

Body Weight

Group

#

#

#

 

#

#

 

#

#

 

#

#

 

Group: 1 Control

0 mg/kg/day

Mean

401.79

13.744

13.33

3.42

3.722

3.680

0.928

1.352

1.334

0.337

0.0217

0.0210

0.0054

SD

18.57

0.881

 

0.20

0.428

 

0.111

0.146

 

0.037

0.0037

 

0.0010

N

10

10

10

10

10

10

10

10

10

10

10

10

10

Group: 2

100 mg/kg/day

Mean

413.45

14.572

13.66

3.52

3.527

3.436

0.854

1.304

1.265

0.315

0.0224

0.0210

0.0054

SD

24.52

1.010

 

0.14

0.299

 

0.071

0.121

 

0.022

0.0032

 

0.0007

N

10

10

10

10

10

10

10

10

10

10

10

10

10

Group: 3

300 mg/kg/day

Mean

385.64

14.826

15.09

dd2

3.83

3.470

3.497

0.903

1.272

1.283

0.332

0.0222

0.0220

0.0058

SD

32.22

2.185

 

0.28

0.287

 

0.079

0.129

 

0.040

0.0033

 

0.0008

N

10

10

10

10

10

10

10

10

10

10

10

10

10

Group: 4

1000 mg/kg/day

Mean

367.01

d1

15.431

d1

16.49

dd2

4.21

3.555

3.661

0.971

1.253

1.298

0.342

0.0222

0.0230

0.0060

SD

28.81

1.129

 

0.20

0.299

 

0.078

0.151

 

0.035

0.0041

 

0.0009

N

10

10

10

10

10

10

10

10

10

10

10

10

10

# [Statistically Analysed]

1 [d - Test: Dunnett 2 Sided p < 0.05] 2 [dd - Test: Dunnett 2 Sided p < 0.01]

 

Table 10. Organ Weights (g) - P Generation Females - Group Mean Values

Sex: Female

Dead

Body Weight

Absolute

Liver Weight

Adjusted

Liver Weight

Liver %

Body Weight

Absolute

Thyroids wt

Adjusted

Thyroids wt

Thyroids %

Body Weight

Group

#

#

#

 

#

#

 

Group: 1 Control

0 mg/kg/day

Mean

261.23

11.405

11.60

4.37

0.0157

0.0159

0.0060

SD

18.65

1.266

 

0.36

0.0033

 

0.0011

N

10

10

10

10

10

10

10

Group: 2

100 mg/kg/day

Mean

267.91

11.783

11.64

4.39

0.0187

0.0186

0.0070

SD

15.92

1.108

 

0.25

0.0031

 

0.0010

N

10

10

10

10

10

10

10

Group: 3

300 mg/kg/day

Mean

268.53

12.153

11.98

4.52

0.0194

d2

0.0192

0.0072

SD

11.51

1.113

 

0.31

0.0033

 

0.0013

N

10

10

10

10

10

10

10

Group: 4

1000 mg/kg/day

Mean

262.23

12.979

dd1

13.12

dd1

4.95

0.0181

0.0183

0.0069

SD

12.13

0.780

 

0.25

0.0032

 

0.0014

N

9

9

9

9

9

9

9

# [Statistically Analysed]

1 [dd - Test: Dunnett 2 Sided p < 0.01] 2 [d - Test: Dunnett 2 Sided p < 0.05]

 

Conclusions:
In this GLP compliant OECD 421 study, a reproduction/ developmental toxicity screening was performed on male and female Crl:WI(Han) mice. The following NOAELs were found:
- Systemic toxicity: 100 mg/kg bw/day based on reduced body weight, increased liver weights and minimal hepatocellular hypertrophy in males.
- Reproductive toxicity: > 1000 mg/kg bw/day
- Developmental toxicity: > 1000 mg/kg bw/day
Executive summary:

In this GLP compliant OECD 421 study, a reproduction/ developmental toxicity screening was performed. Forty male and 40 female rats of the Crl:WI(Han) strain were allocated to the study. Groups of 10 males and 10 females were dosed with 0 (vehicle), 100, 300 or 1000 mg/kg/day test substance, once daily, by oral gavage at a dose volume of 10 mL/kg body weight using 1% aqueous carboxymethylcellulose as vehicle. Males were dosed for 14 days before and during pairing and until the day before necropsy (47 days) and the females were dosed for 14 days before pairing, during pairing and then until Day 3 of lactation (approximately 42 days). All animals were examined for effects on general condition, body weight and food intake. During the pairing period vaginal smears were taken daily until sperm were found in the smear. The females were allowed to litter and rear their offspring to Day 4 of age. A macroscopic necropsy was performed on all parental animals and F1 generation pups. For the parental males and females a selection of organs were weighed, fixed and examined microscopically.

There were no deaths considered to be related to treatment with the test item and no test-item related clinical signs. Throughout the dosing period, males given 300 mg/kg/day or 1000 mg/kg/day showed reduced body weight gain, compared with Controls. There was no effect on body weight or body weight gain for females at any dose level or for males given 100 mg/kg/day. There was no effect on food intake for males or females at any dose level. Fertility and mating performance were unaffected by treatment with the test substance and there were no effects on pregnancy parameters. Males given 300 mg/kg/day and both sexes given 1000 mg/kg/day showed increased liver weights adjusted for body weight. Minimal hepatocellular hypertrophy was noted in males given 300 mg/kg/day and in both sexes given 1000 mg/kg/day. Minimal thyroid follicular hypertrophy was also seen in males given 300 or 1000 mg/kg/day. There was no effect of maternal treatment with the test substance on pup body weights to Day 4 of age. Pup survival was similar across all groups including the Controls.

In conclusion, the test substance administered once daily by oral gavage to male and female rats at doses of 0, 100, 300 or 1000 mg/kg/day was well tolerated at all dose levels. The No Observed Adverse Effect Level (NOAEL) for male and female reproductive performance was 1000 mg/kg/day. The NOAEL for parental toxicity was 100 mg/kg/day. Reduced body weight and thyroid follicular hypertrophy were observed at the higher dose levels in males only. Increased liver weight in association with minimal hepatocellular hypertrophy was observed at 300 (males only) and 1000 mg/kg/day, in both sexes with males being more severely affected than females. The NOAEL for toxicity to the offspring was 1000 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP compliant OECD 421 study
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

See additional information in the Fertility section.

Justification for classification or non-classification

Based on the absence of adverse effects in the reproduction/developmental toxicity screening test in rats, classification is not warranted for developmental toxicity in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Additional information