Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010 - 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011
Reference Type:
other: study report amendment
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Version / remarks:
adopted May 1983
Deviations:
no
Principles of method if other than guideline:
In addition, the study was enhanced by parameters which are part of the following test guidelines:

• Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Part B: Methods for the determination of toxicity and other health effects: Two-Generation Reproduction Toxicity Study; Official Journal of the Union, No. L 142, pp. 355-364
• EPA Health Effects Test Guidelines, OPPTS 870.3800: Reproduction and Fertility Effects (Aug 1998)
• OECD Guidelines for Testing of Chemicals; No. 416 (22 Jan 2001)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Solid/ light yellow
- Analytical purity: 99.9%
- Lot/batch No.: 37973FC4AA
- Storage condition of test material: Room temperature, under light exclusion

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: (P) 28 (±1) days at arrival from the breeder
- Weight at study initiation: (P) Males: 128.6 g - 155.8 g; Females: 103.1 g - 124.3 g
- Housing: individually in Makrolon type M III cages, with the following exceptions: during overnight matings, male and female mating partners were housed together in Makrolon type M III cages; and pregnant animals and their litters were housed together until PND 21 (end of lactation).
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland;ad libitum
- Water (e.g. ad libitum): Drinking water was supplied from water bottles (ad libitum)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance preparations in Propylene glycol were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration solutions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with Propylene glycol and subsequently thoroughly mixed with a homogenizer. During administraton the preparations were kept homogeneous with a magnetic stirrer.
Details on mating procedure:
At least 74 days after the beginning of treatment, males and females from the same dose group were mated
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight ratio for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of gestation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in Propylene glycol for a period of 7 days at room temperature were carried out before the study was initiated.
The samples, which were taken for the concentration control analyses at the beginning of the administration period, were also used to verify the homogeneity for the samples of the low and the high concentrations (30 and 300 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running.
Duration of treatment / exposure:
F0
males: 110 d = ca. 16 weeks
females: 126 d = 18 weeks
Frequency of treatment:
once daily at approximately the same time in the morning
Details on study schedule:
- Age at mating of the mated animals in the study: 109 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Outcome of dose-range-finding study (28 days)

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each animal and reported on a weekly basis.

BODY WEIGHT: Yes
- Time schedule for examinations: In general, the body weight of the male and female parental animals and of the F1 rearing animals was determined on the first day of test substance administration or the first collective weighing day (day 0), respectively, and then once a week at the same time of the day (in the morning).
The following exceptions are notable for the female animals:
• The F0 generation parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 1, 4, 7, 14 and 21.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Generally, food consumption was determined once a week for male (until 10th premating week) and female (until weaning) F0 parental animals and F1 rearing animals, with the following exceptions:
• During pregnancy, food consumption of the F0 females with evidence of sperm was determined weekly for GD 0 - 7, 7 - 14 and 14 - 20.
• During lactation, food consumption of the F0 females, which gave birth to a litter was determined for PND 1 - 4, 4 - 7, 7 - 14, and 14 - 21.
Food consumption was not determined during the mating period as well as for females without positive evidence of sperm and females without litter.
Oestrous cyclicity (parental animals):
Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats for a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.
Sperm parameters (parental animals):
not studied
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible)

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths (pups, which died before this initial examination, were defined as stillborn pups, live births), postnatal mortality, macroscopic (external and visceral) examination. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were further evaluated on a case-by-case basis (e.g., histopathological evaluation or special staining), depending on the findings noted.
The pups were weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7, 14 and 21.
Postmortem examinations (parental animals):
All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia and were exsanguinated. The animals were necropsied and assessed by gross pathology, with particular attention given to the reproductive organs. Animals which died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after their death and assessed by gross pathology.

Organ weights
The following weights were determined in all parental animals sacrificed on schedule:
Anesthetized animals; Liver; Kidneys; Adrenal glands; Testes; Epididymides; Cauda epididymis; Prostate; Seminal vesicles including coagulation glands; Ovaries; Uterus; Spleen; Brain; Pituitary gland; Thyroid glands (with parathyroid glands)

Organ/Tissue fixation
The following organs or tissues of the F0 generational parental animals were fixed in 4% buffered formaldehyde solution or modified Davidson’s solution:
Vagina; Cervix uteri; Uterus; Ovaries (fixed in Davidson’s solution); Oviducts; Testes (fixed in Davidson’s solution); Epididymides (fixed in Davidson’s solution); Seminal vesicles; Coagulation glands; Prostate; Pituitary gland; Adrenal glands; All gross lesions; Liver; Kidneys; Spleen; Brain; Thyroid glands (with parathyroid glands); Stomach
Ovaries, testes and epididymides of animals that died or were sacrificed intercurrently were fixed in 4% buffered formaldehyde solution.

Histopathology was performed for reproductive organs and organs that were affected by weight changes and/or gross lesions (liver, thyroid, kidney, adrenal glands, stomach).
Postmortem examinations (offspring):
Pup organ weights
After the scheduled sacrifice the brain, spleen and thymus of 1 pup/sex and litter from the F1 pups were weighed. For the calculation of the relative organ weights, the inlife pup weights determined on PND 21 were used.

Pup necropsy observations
All pups with scheduled sacrifice (i.e. pups culled on PND 4 or sacrificed on PND 21) were examined externally and eviscerated; their organs were assessed macroscopically.
All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

Statistics:
Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), estrous cycle duration, number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means
Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions
Proportions of affected pups per litter with necropsy observations: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
Organ weights (absolute and relative; parentals and pups): Non-parametric one-way analysis using KRUSKAL-WALLIS-test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON-test (two-sided) for the equal medians.
Reproductive indices:
For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:
Male mating index (%) = (number of males with confirmed mating*/number of males placed with females) x 100
* defined by a female with vaginal sperm or with implants in utero
Male fertility index (%) = (number of males proving their fertility*/ number of males placed with females) x 100
* defined by a female with implants in utero

For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:
Female mating index (%) = (number of females mated*/number of females placed with males) x 100
* defined as the number of females with vaginal sperm or with implants in utero
Female fertility index (%) = (number of females pregnant*/ number of females mated**) x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero
Gestation index (%) = (number of females with live pups on the day of birth/ number of females pregnant*) x 100
* defined as the number of females with implants in utero
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters according to the following formula:
Live birth index (%) = (number of liveborn pups at birth/ total number of pups born) x 100
The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:
Postimplantation loss (%) = ((number of implantations – number of pups delivered)/ number of implantations) x 100

Sex ratio
Sex ratio = (number of live male or female pups on PND 0 and 21/ number of live male and female pups on PND 0 and 21) x 100
Offspring viability indices:
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays.
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1 - 4, 5 - 7, 8 - 14 and 15 - 21 (lactation period) were determined; however, pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation days 4, 7, 14, and 21. Furthermore, viability and lactation indices were calculated according to the following formulas:
Viability index (%) = (number of live pups on day 4* after birth/number of live pups on the day of birth) x 100
* before standardization of litters (i.e. before culling)
Lactation index (%) = (number of live pups on day 21 after birth/ number of live pups on day 4* after birth) x 100
* after standardization of litters (i.e. after culling)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All dosed males and all dosed females showed salivation after treatment (from study week 5 onwards). Although all treated animals were affected at least once during the study the daily incidence for salivation was higher in the mid and high dose group than in the low dose group.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male animal of the 100 mg/kg bw/d test group was found dead in study week 10, but did not display any histopathological findings. One female animal of 30 mg/kg bw/d test group which was sacrificed moribund in study week 15 showed edema in anal and/or genital region, poor general state, and abdominal distension
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test substance-related changes of mean body weights or body weight gain were noted for all treated males during the entire study. Because of it´s isolated occurrence a statistically significantly decreased body weight gain during weeks 12 - 13 in the high-dose males is considered to be incidental.
No test substance-related changes of mean body weights or body weight gain were noted for all treated females during premating. Body weights of high-dose parental females was consistently and statistically significantly lower than the concurrent control on GD 7 - 20 (up to 7%) and during the whole lactation period (up to 8%).
The statistically significantly increased mean body weights/body weight gain in the mid-dose females on study week 6 and week 10 and during study weeks 0 - 1 and weeks 0 – 10 were considered as spontaneous in nature, as was the statistically significantly lower mean body weight gain in females of all dose groups during PND 4 - 7.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Neither males of all dose groups nor females of low- and mid-dose groups showed any test substance-related changes of food consumption during the entire study.
Food consumption of the high-dose F0 females was also unchanged during premating and gestation. The statistically significantly decreased or increased food consumption in the high and mid-dose females during during study week 0 - 1, study weeks 2 - 3 and 4 - 5 was considered as spontaneous in nature.
However, in the high dose test group food consumption was statistically significantly below control during the whole lactation period (up to 20%).
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related findings were observed in reproductive organs of males (testes, epididymides, prostate, seminal vesicles and coagulating glands) and females (ovaries, oviducts, uterus, cervix and vagina) in the high dose test group.
Of the stomach only the gross lesions were examined histopathologically. All animals were investigated for liver, kidney, thyroid and adrenal glands.

Minimal to slight mucosal hyperemia was seen in the glandular stomach in males (11/15) of the high dose test group. In males and females of the mid dose test group as well as in females of the high dose test group, hypermemia was minimal in incidence and grading.

In the liver, a dose-dependent weight increase observed in males and females, starting from test group 02 (100 mg/kg bw/d) was regarded as treatment-related. In test group 03 (300 mg/kg bw/d), weight increases (absolute / relative: 126% / 134% in males; 144% / 150% in females), which were above historical control values (abs. / rel.: 9.615 g / 2.53% in males, 7,811 g / 3.28% in females), correlated with minimal to slight hypertrophy of central to midzonal hepatocytes and organ enlargement. No histopathological correlate was seen in males and females of test group 02 (100 mg/kg bw/d) that could justify the liver weight increase (relative: 110% in males; absolute / relative: 114% / 112% in females), which was also above the maximal historical values. Although no signs of hepatotoxicity were seen in males and females test group 3 (300 mg/kg bw/d), based on the magnitude of the relative liver weight deviations exceeding 30% of the control and the lack of hepatic clinical chemical data, the findings were regarded as adverse, whereas findings in male and females of test group 02 (100 mg/kg bw/d) were regarded as treatment-related and adaptive. The green brown or dark brown discoloration noted at gross pathology was consistent with minimal brown gold pigment storage in central hepatocytes at 300 mg/kg bw/d but not at 200 mg/kg bw/d. This finding was minimal and was considered to be treatment-related but not adverse. No treatment-related findings were seen in the liver of males and females of test group 01 (30 mg/kg bw/d).

Findings on kidney, thyroid and adrenal glands were non-adverse and are described in detail (including tables) in the endpoint study record for repeated-dose toxicity.
All other findings noted were either single observations, or were biologically equally distributed between control and treated rats. All of them were considered to be incidental and/or spontaneous in origin.

One moribund sacrificed female of the low dose test group showed a severe to moderate subacute inflammation in the uterus, vagina and ureters. The regional renal and iliac lymph nodes were found with severe lymphoreticulo-cellular hyperplasia in response to the inflammation. These findings explained the moribund state of this animal. No histopathological findings were seen in one male of the mid dose test group that could explain its death.

All non pregnant females as well as their male mating partners did not show relevant histopathological findings that could explain the impaired fertility in these animals.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 3 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration in the different test groups was similar: 4.1 days in control and mid-dose groups, 4.0 in low and high-dose groups.
Reproductive performance:
no effects observed
Description (incidence and severity):
The male and female mating indeces was 100% in all test groups. The mean duration until sperm was detected (GD 0) varied between 2.3 and 3.0 days without any relation to dosing.
Three sperm positive females of both the 300 and 100 mg/kg bw/d test groups, one sperm positive female of the 30 mg/kg bw/d test groups and two sperm positive females of the control group did not deliver F1 pups.
The fertility index ranged between 85% (high dose group) and 100%. All respective values, including the apparently lower high-dose value, are within the range of the historical control data of the test facility. Thus, all values reflect the normal range of biological variation inherent in the strain of rats used for this study. Furthermore, the apparently infertile male rats did not show histopathological findings that could explain infertility. Additionally, there were no corroborative histopathological findings in the sexual organs of the non-pregnant females.
Two high dosed dams showed the finding ‘no more pups alive’.
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls.
The mean duration of gestation was similar in all test groups: 22.0 days for the mid-dose group, 22.1 days for control and high-dose group and 22.2 days for the low-dose group.
The gestation index was 95% in control, 100% in the low and high-dose group, and 85% in the mid-dose group.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (11.6 / 10.9 / 10.6 and 10.6 implants/dam in test groups 0, 30, 100 and 300 mg/kg body weight/day). Postimplantation loss was not affected by the treatment being 17.7 / 8.5 / 20.1 and 12.8% in test groups 0, 30, 100 and 300 mg/kg body weight/day.
The number of dams with stillborn pups and the number of stillborn pups were statistically significantly increased in the mid- and high-dose groups. This affected the live birth index which was statistically significantly decreased for the mid-dose (about 3% below control) and high-dose dams (about 6% below control). No effect on live birth index was observed in the low-dose group.
The total mean of delivered pups per group showed no statistically significant differences between treated and control groups.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No effects at dose level

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
The F1 generation pups did not display any clinical signs until weaning.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The number of liveborn pups was statistically significantly lower, but within the historical control range in the mid-dose group (about 5% below control) and statistically significantly lower and outside the historical control range in the high-dose group (about 21% below control). The number of liveborn pups was comparable to the control in the low dose group. These values correspond to increased or unchanged numbers of stillborn pups.
Pup mortality was statistically significantly increased in the high-dose group, as 18 pups died and 5 pups were cannibalized in this group. Almost all of these pups died within 4 days after birth. (In the high and mid dose group, each one pup died after day 4 which is considered incidental).
No such findings were observed in the low- and mid-dose group.
Thus, the viability index indicating pup mortality during early lactation (PND 0 - 4) was reduced in the high-dose group (86% vs. 100% in control). This parameter was unchanged in the low- and mid-dose group. The lactation index showed no effects for all dose groups.
The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 21 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of all high-dose F1 pups were consistently and statistically significantly below control during the entire lactation period (up to 24% when both sexes combined). Body weight gain of high-dose F1 pups was affected in the same way; the difference to the control was up to 36% (both sexes combined). This is considered to be related to the lower food consumption and body weight gain of lactating females of the high dose group.
No test compound-related influence on F1 pup body weights or weight gains was noted in all pups of the mid and low-dose groups.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Absolute and relative organ weights were comparable to the control group in the low and mid dose groups. In the high dose group, absolute weights of the brain, thymus and spleen were significantly reduced to 95, 73 and 70% of the weights of the control group, respectively. Expressed as relative weights, the respective values were 121, 90 and 87%, respectively.
The pattern of the changes indicates that they were secondary to the decreased pup body weights. There was no difference between the sexes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
F1 pups in all groups showed spontaneous findings at gross necropsy, such as post mortem autolysis, hemorrhagic thymus, diaphragmatic hernia, hydronephrosis, hydroureter and small testis. The apparently higher number of pups with post mortem autolysis in the high dose test group resulted from the overall higher number of decedents in this dose group. No treatment-related findings were noted upon gross pathology.
Histopathological findings:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
reproductive performance and developmental toxicity
Generation:
F1
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Mean maternal body weights during gestation

Test group

(mg/kg bw/d)

 

0

Control

1

(30)

2

(100)

3

(300)

Day 0

MEAN

218.10

217.80

224.90

209.20

 

S.D.

10.80

14.75

15.03

12.46

 

N

19

19

20

17

Day 7

MEAN

242.40

241.40

247.90

229.6*

 

S.D.

11.82

14.12

16.62

13.81

 

N

19

19

20

17

Day 14

MEAN

265.80

265.60

270.60

250.6*

 

S.D.

15.12

14.98

16.30

18.58

 

N

19

19

20

17

Day 20

MEAN

315.80

311.70

314.50

292.7*

 

S.D.

23.46

22.96

26.97

28.58

 

N

19

19

20

17

Dunnett-test (two-sided) *p<=0.05

Mean maternal body weights during lactation

Test group

(mg/kg bw/d)

 

0

Control

1

(30)

2

(100)

3

(300)

Day 0

MEAN

248.8

246.4

252.4

232.0*

 

S.D.

14.4

12.46

15.32

18.17

 

N

18

19

17

17

Day 1

MEAN

244.3

246.2

252.6

229.4**

 

S.D.

15

11.89

15.23

13.44

 

N

18

19

17

17

Day 4

MEAN

255.2

259.1

263.3

240.6*

 

S.D.

15.94

11.81

16.1

14.08

 

N

18

19

17

17

Day 7

MEAN

266.2

264.3

269.9

248.0**

 

S.D.

16

11.4

15.16

14.6

 

N

18

19

17

17

Day 14

MEAN

280.2

278.2

285.9

256.7**

 

S.D.

18.3

13.76

15.54

18.23

 

N

18

19

17

17

Day 21

MEAN

273

274.5

278.6

260.3*

 

S.D.

11.67

13.25

10.02

17.71

 

N

18

19

17

17

Dunnett-test (two-sided) *p<=0.05, **<=0.01

Mean maternal body weight changes during gestation and lactation (g)

Test group

(mg/kg bw/d)

0

control

1

(30)

2

(100)

3

(300)

Gestation Day 0 to 7

MEAN

24.30

23.60

23.00

20.40

 

S.D.

5.14

4.87

6.29

6.43

 

N

19

19

20

17

Gestation Day 7 to 14

MEAN

23.40

24.20

22.70

21.00

 

S.D.

6.63

7.35

6.62

6.76

 

N

19

19

20

17

Gestation Day 14 to 20

MEAN

50.00

46.10

43.90

42.10

 

S.D.

12.03

12.21

19.87

14.98

 

N

19

19

20

17

Gestation Day 0 to 20

MEAN

97.70

93.90

89.60

83.50

 

S.D.

19.66

19.09

25.51

22.57

 

N

19

19

20

17

Lactation Day 0 to 1

MEAN

-0.5

-0.2

0.3

-2.6

 

S.D.

5.69

8.75

7.51

9.09

 

N

18

19

17

17

Lactation Day 1 to 4

MEAN

11

12.9

10.7

11.2

 

S.D.

7.87

5.14

7.14

5.9

 

N

18

19

17

17

Lactation Day 4 to 7

MEAN

11

5.2**

6.5*

7.4

 

S.D.

6.23

4.06

4.56

5.8

 

N

18

19

17

17

Lactation Day 7 to 14

MEAN

13.9

13.9

16.1

8.7

 

S.D.

8.35

8.8

9.24

8.17

 

N

18

19

17

17

Lactation Day 14 to 21

MEAN

-7.2

-3.7

-7.3

3.6**

 

S.D.

10.86

7.86

9.16

6.58

 

N

18

19

17

17

Lactation Day 0 to 21

MEAN

28.2

28.1

26.3

28.3

 

S.D.

10.94

11.79

9.77

13.49

 

N

18

19

17

17

 

Female reproduction and delivery data – group means

Dose group

Gestation index (%)

Females with stillborn pups

N (%)

Live birth index (%)

Stillborn pups  

N (%)

Viability index

N (%)

Lactation index

N (%)

Control

95

0 (0)

100

0 (0)

194 (100)

134 (100)

30 mg/kg

100

1 (5.3)

99

2 (1.0)

185 (98)

139 (100)

100 mg/kg

85

5 (29)*

97

6 (3.2)*

181 (98)

129 (99)

300 mg/kg

100

8 (47)*

94

9 (5.6)*

131 (86)*

103 (99)

Historical data (%)

91 - 100

---

(95 – 100)

(0 – 4.5)

(94 – 100)

(94 – 100)

*p<=0.05

Maternal and pup weights – group means

Dose group

Terminal BW (females, day 21p.p.)

Pups Day 1 (M+F)

(g)

PupsDay 4 (M+F)

(g)

PupsDay 7 (M+F)

(g)

Pups Day 14 (M+F)

(g)

PupsDay 21 (M+F)

(g)

Control

273.0

6.3

9.3

14.7

29.3

46.6

30 mg/kg

274.5

6.3

9.5

15.0

29.5

46.3

100 mg/kg

278.6

5.9

8.8

14.0

28.1

44.3

300 mg/kg

260.3*

5.5*

7.8*

11.1*

22.6*

36.9*

Historical data

226.7 – 307.7

5.8 – 6.9

8.6 – 10.6

13.1 – 17.3

25.5 – 33.4

41.3 – 53.7

*p<=0.05

Applicant's summary and conclusion

Conclusions:
The NOAELs for both parental animals and offspring were determined to be NOAEL (maternal toxicity) 100 mg/kg bw/day, NOAEL (fertility) 100 mg/kg bw/day, and NOAEL (development) 100 mg/kg bw/day under the conditions of the test.
Executive summary:

In this guideline (OECD 415) conducted with GLP certification, the parental fertility NOAEL was determined to be 100 mg/kg bw/day. The test was conducted on rats (20 per sex, per dose level), dose levels were set at 0, 30, 100, and 300 mg/kg bw/day. The test material was administered by gavage. Clinical effects (salivation) were noted in all test groups. Treatment related body weight, food consumption, organ wieght, histopathological, and fertility effects were observed in parental animals at the 300 mg/kg bw/day group. Mortality and body weight gain effects were observed in the offspring of the 300 mg/kg bw/day group.

The study provides clear evidence of adverse effect on sexual function and fertility in parental animals, as well as developmental effects (mortality) in offspring. In the absence of any information on mechanistic effects, the test material is considered to meet the EU Classification, Labelling, and Packaging (CLP) regulation (1272/2008) criteria for reproductive toxicity.