Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Study conducted to recognised training guidelines with GLP

Link to relevant study records
Reference
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
Qualifier:
according to guideline
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. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system 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
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
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)
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)
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: 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.
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
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
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
Reproductive effects observed:
not specified

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

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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a GLP conform 1-generation study according to OECD guideline 415, the test item was administered daily as preparation in propylene glycol to groups of 20 male and 20 female healthy young Wistar rats (F0 parental generation) by stomach tube at doses of 30, 100 and 300 mg/kg body weight/day (BASF 2011). Control animals were dosed daily with the vehicle only (propylene glycol). At least 74 days after the beginning of treatment, F0 animals were mated to produce a litter (F1 rearing animals). Mating pairs were from the same dose group.

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. Food consumption of the F0 parental generation animals was determined once weekly (F0 males and F0 females until 10th premating week) and usually for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4, 4 - 7, 7 - 14 and 14 - 21. In general, body weights of F0 parental generation animals were determined once weekly. However, during gestation and lactation F0 females were weighed on gestation days (GD) 0, 7, 14 and 20 and on postnatal days (PND) 0, 1, 4, 7, 14 and 21. Estrous cycle data were evaluated for F0 generation females over a three week period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice. The F1 pups were sexed on the day of birth (PND 0) and were weighed on the first day after birth (PND 1) as well as on PND 4, 7, 14 and 21. Their viability was recorded. At necropsy, all pups were examined macroscopically (including weight determinations of brain, spleen and thymus in one pup/sex/litter). All F0 parental animals were assessed by gross pathology (including weight determinations of several organs) and subjected to histopathological examination, special attention being paid to the organs of the reproductive system. Histopathology was also performed for liver, kidney, adrenal gland and thyroid.

Parental toxicity was noted at the high dose group. It consisted mainly on reduced food consumption and body weight gain for females during lactation and on a strong liver elargement accompanied by histopathology changes. The NOAEL for systemic toxicity was 100 mg/kg bw. Details are described in the section for developmental toxicity as pup development was adversely affected in this study.

No adverse effects on fertility parameters were noted in this study. There were no indications from clinical examinations as well as gross and histopathology, that the test substance adversely affected the fertility of the F0 parental animals up to a dose of 300 mg/kg bw/day. Estrous cycle data, mating behavior, conception, as well as sexual organ weights and gross and histopathological findings of these organs were comparable between the rats of all test groups and ranged within the historical control data of the test facility. Most of the F0 parental animals proved to be fertile. Failure of pregnancy (infertility) in one control female and in four test substance-treated females (one low-dose and three high-dose rats) could not be attributed to the treatment by gross and histopathological examinations of the respective animals of both genders.

As part of the subacute oral toxicity studies, some parameters related to fertility were investigated.

During the 28-day dose-range finding study with each 5 male and female rats (NOTOX 2009), histopathology of testes and epididymides was performed for control and high dose group males; slides of the testes were prepared to examine staging of spermatogenesis. Sperm motility was analyzed for all males of the control group, the intermediate dose group (100 mg/kg bw) and the high dose group (initially 500 mg/kg bw, later reduced to 250 mg/kg bw). As a result, no microscopic findings were noted on epididymides. One animal of the high dose group showed seminiferous atrophy. Sperm motility analyses revealed no abnormalities. The staging of spermatogenesis suggested normal spermatogenesis; all stages were present. No macroscopic findings on reproductive organs were noted. Weights of reproductive organs were not determined.

During the 28-day oral toxicity study performed with dose levels of 10, 50 and 500 mg/kg bw (Hazelton 1989), no macroscopic abnormalities were noted for reproductive organs. As a result, their weights were not determined and no histopathology was performed.

Gonad weights were determined during the 14-day range-finding study performed with 100, 300, 1000 and 3000 mg/kg bw (Hazelton 1989a). Incidences of mortality occurred in females at 1000 and 3000 mg/kg bw. There were no changes in relative organ weights of gonads.

Short description of key information:  In a 1-generation study according to OECD guideline 415 and GLP requirements, rats received doses of 30, 100 and 300 mg/kgbw/d per gavage (BASF SE 2011). The NOAEL (no observed adverse effect level) for fertility was 300 mg/kg bw/d. The NOAEL for general, systemic toxicity of the test substance was 100 mg/kg bw/d for males and females based on adverse effects on liver and for females only reduced food consumption and body weight gain at next higher dose level.

Effects on developmental toxicity

Description of key information
A one-generation study in rats is available (OECD 415, GLP). The NOAELs was found to be 100 mg/kg bw/d, based on reduced live birth index, pup mortality and reduced pup weights at next higher dose level of 300 mg/kg bw. A specific study for teratogenicity is not available.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Data waiving:
other justification
Justification for data waiving:
other:
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
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

In a GLP conform 1-generation study according to OECD guideline 415, the test item was administered daily as preparation in propylene glycol to groups of 20 male and 20 female healthy young Wistar rats (F0 parental generation) by stomach tube at doses of 30, 100 and 300 mg/kg body weight/day (BASF 2011). Control animals were dosed daily with the vehicle only (propylene glycol). At least 74 days after the beginning of treatment, F0 animals were mated to produce a litter (F1 rearing animals). Mating pairs were from the same dose group.

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. Food consumption of the F0 parental generation animals was determined once weekly (F0 males and F0 females until 10th premating week) and usually for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4, 4 - 7, 7 - 14 and 14 - 21. In general, body weights of F0 parental generation animals were determined once weekly. However, during gestation and lactation F0 females were weighed on gestation days (GD) 0, 7, 14 and 20 and on postnatal days (PND) 0, 1, 4, 7, 14 and 21. Estrous cycle data were evaluated for F0 generation females over a three week period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice. The F1 pups were sexed on the day of birth (PND 0) and were weighed on the first day after birth (PND 1) as well as on PND 4, 7, 14 and 21. Their viability was recorded. At necropsy, all pups were examined macroscopically (including weight determinations of brain, spleen and thymus in one pup/sex/litter). All F0 parental animals were assessed by gross pathology (including weight determinations of several organs) and subjected to histopathological examination, special attention being paid to the organs of the reproductive system. Histopathology was also performed for liver, kidney, adrenal gland and thyroid.

Effects on the F0 generation

Clinically, toxicity was noted in the F0 females at 300 mg/kg bw/d. Food consumption was reduced during lactation, body weights /body weight gain were consistently reduced during gestation and lactation.

All high-dose as well as most mid-dose and many low-dose animals of both sexes showed transient salivation for a few minutes immediately after each treatment. This was likely to be induced by the unpleasant taste of the test substance and/or by local irritation of the upper digestive tract. It is neither considered to be a sign of systemic toxicity nor as adverse.

Pathology confirmed adverse clinical observations were likely to be subsequent to local irritant effects of the test substance in the fore- and glandular stomach. Also, reduced food consumption and weights/body weight gain in females mainly during lactation may have been secondary to these local effects.

In the glandular stomach of males and females of test groups 2 (100 mg/kg bw/d) and 3 (300 mg/kg bw/d), minimal to slight mucosal hyperemia correlated with “red focal discolorations”. Males were more affected than females and showed a clear dose relationship in test group 3 (300 mg/kg bw/d). Mucosal hyperemia was attributed to a local effect due to treatment but was regarded as not adverse.

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 / 2.529% 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 03 (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).

In the kidneys of male animals, although control weights were minimally above the maximal historical control values, the relative weight increase of 109% and 113% observed respectively in test groups 02 and 03 (100 and 300 mg/kg bw/d) were regarded as treatment-related and were associated with histopathological findings. These showed a dose-dependent increase of eosinophilic droplets in the proximal convoluted tubules. The eosinophilic droplets displayed at the Chromotrope-Aniline Blue stain (CAB) a histomorphologic pattern and tubular distribution characteristic of α2u-globulin (Hard, 2008). α2u-globulin is a male and rat specific poor soluble protein synthesized in the male rat liver, filtered by the glomerulus and reabsorbed in the S2 segment of the proximal tubules where it is slowly hydrolyzed by lysosomal digestion. Reversible binding of the test-substance or their metabolites to α2u-globulin decreases the effectiveness of its lysosomal digestion, resulting in strong accumulation of this protein that can be visible in form of characteristic eosinophilic droplets. This may lead to increased incidence or severity of chronic progressive nephropathy, including increased numbers of cortical basophilic tubules, lymphoid cell infiltrates and tubular dilation as seen in test group 02 and 03 males. Although the increase of eosinophilic droplets and its associated findings is considered treatment-related, this finding does not represent a risk for humans since they do not synthesize this protein (Durham and Swenberg, 2013). No histopathological correlate was seen in the kidneys of males in test group 01 (30 mg/kg bw/d) that could explain the minimal relative kidney weight increase (105%), which was regarded as incidental.

In females of test group 03 (300 mg/kg bw/d), although the weights were significantly increased (absolute; 109%, relative: 114%) no histopathological correlate could be detected. Nevertheless, since the absolute (1.946 g) and relative (0.876%) weights were slightly above the maximal historical control values (absolute / relative: 1.865 g / 0.771%), this change was regarded as treatment-related but not adverse. No treatment-related findings were seen in the kidneys of test group 01 and 02 (30 and 100 mg/kg bw/d) females. No histopathological correlate was found for the green brown discoloration observed macroscopically in the kidneys of male and female animals of test groups 02 and 03 (100 and 300 mg/kg bw/d). Therefore, this change was regarded as treatment-related but not adverse.

In the thyroid gland, a dose-dependent hypertrophy and / or hyperplasia of the follicular epithelium started to be observed from test group 02 (100 mg/kg bw/d) in males (minimal) and females (minimal to slight). These changes might be associated with an increased thyroxin catabolism occurring in the liver due to hepatic enzyme induction. This well-known phenomenon is characteristic for rodents (Curran et al., 1991), and there is no convincing evidence that humans exposed to chemicals that induce hepatic microsomal enzymes have increased risk for any thyroid gland disorder (Capen, 1997). In males, organ weights including controls were minimally above the maximal historical control values (abs.: 29.000 mg / rel.: 0.008%); however, histopathology was not associated to statistically increased weights. In females, although a significant absolute and relative thyroid weight increase was seen at the high dose (300 mg/kg bw/d), the values were within the historical control range (abs. / rel.: 21.957 g / 0.01%). Thus, the thyroid gland findings in test group 2 and 3 (100 and 300 mg/kg bw/d) were regarded as treatment-related and not adverse. No treatment-related findings were seen in the thyroid gland of males and females of test group 01 (30 mg/kg bw/d).

The adrenal gland of females showed a dose-dependent weight increase starting from test group 02 (100 mg/kg bw/d). In test group 03 (300 mg/kg bw/d), cortical cells with condensed eosinophilic cytoplasm devoid of lipid vacuoles in the zona fasciculata correlated with significantly increased organ weights (absolute/ relative: 122% / 127%). These (abs. / rel.: 94.9 mg / 0.042%) were above the maximal historical control values (abs.: 85.7 mg; rel.: 0.035%). These findings may represent ACTH-induced depletion related to stress (Hamlin II and Banas, 1990). In test group 02 (100 mg/kg bw/d), the weight of the adrenal glands was statistically increased (abs. / rel.: 115% / 113 %) and above the maximal historical control values (abs. / rel.: 89.4 mg / 0.038%) but without any histopathological correlate. Nevertheless, all these findings in test group 02 and 03 females were interpreted as treatment-related, but secondary adaptive. No treatment-related findings were seen in females of test group 01 (30 mg/kg bw/d).

No histopathological correlate was found in the adrenal glands of male animals of test group 03 (300 mg/kg bw/d), which had a statistically significant relative glandular weight increase (112%) but below the maximal historical control values (0.019%). Therefore, the adrenal gland weight increase in males of the high dose group was regarded as incidental and not related to treatment.

In conclusion, the test substance administered at dose levels of 30, 100 and 300 mg/kg bw/d to parental F0 male and female rats led to a minimal statistically significant terminal body weight decrease in females treated with 300 mg/kg bw/d and on adverse effects on liver for both sexes at this dose level.

Effects on offspring

Mid- and high-dose females had a significantly increased number of stillborn pups and, consequentially, a decreased live birth index indicating an adverse effect of the test compound on reproductive performance. However, for the mid-dose group both parameters were well within the historical range of the test facility, in this case the apparent reduction of the live birth index may have been an incidental finding.

For all liveborn pups of the F0 parents, no test substance-induced signs of developmental toxicity were noted at dose levels as high as 100 mg/kg bw/d. Postnatal survival as well as post-weaning development of the offspring of these test groups until weaning remained unaffected by the test substance. Furthermore, clinical and/or gross necropsy examinations of the F1 revealed no adverse findings.

Pup mortality was statistically significantly increased and pup body weights were statistically significantly reduced in the high-dose group (300 mg/kg bw/d). The number of live pups per litter as well as the total number of pups explicitly discriminates the high-dose group from the lower dose groups and controls. Pup mortality was seen within 4 days after birth. Both findings are regarded as treatment-related developmental toxicity and slight delay of postnatal development. However, it should be noted that the respective findings were seen especially in litters where the dams prenatally and postnatally showed a clear reduction of body weights/body weight gain and/or their food intake was affected. These findings were noted exclusively in the high-dose group. In the high-dose female population, 6 dams that had a body weight gain (BWG) below the group mean value consisted of the only two dams with total litter loss and three dams with small litter (n= 3, 4, 5 or 6). The small litter size itself may well have contributed to the lower body weight of the dams. However, the study design does not allow for quantitatively discriminating of parental versus litter toxicity. The group mean value for body weight gain at the end of the gestation period was 83.5 g (approx. - 15% compared to controls, value= 97.7 g). Several of the six animals with BWG below average have individual BWG values that amount to only 50 to 70% of the group mean. With this reduction, the internationally acknowledged criteria for the MTD (-10%) is exceeded.

Secondary to the reduced pup body weights, lower weights of spleen and thymus as well as lower absolute and higher relative brain weights were noted in these offspring, these effects were not regarded as adverse or toxicologically relevant findings. Any other developmental parameters such as postnatal survival from PND 4 remained unaffected by the test substance.

Justification for classification or non-classification

In a 1-generation study according to OECD guideline 415 and GLP requirements, rats received doses up to 300 mg/kg bw/d per gavage (BASF 2011). The NOAEL (no observed adverse effect level) for fertility was 300 mg/kg bw/d for the parental rats, the highest dose tested. The repeat-dose toxicity study in rats did not cause adverse effects to the reproductive organs of male and female rats. Therefore, untoward alteration of fertility is considered to be unlikely. It is concluded that the test substance is not subject to classification for fertility. The NOAEL for general, systemic toxicity of the test substance was 100 mg/kg bw/d for males and females based on adverse effects on liver, and in the case of females also based on reduced food consumption and body weight gain mostly during lactation. The NOAEL for reproductive performance and developmental toxicity in the F1 progeny of the test substance treated groups was determined at 100 mg/kg bw/d, based on reduced live birth index, pup mortality and reduced pup weights at the higher dose level. Toxicity to the offspring was observed at a dose that elucidated maternal toxicity. It is acknowledged that the development of the offspring throughout gestation and during the early postnatal stages can be influenced by toxic effects in the mother either through non-specific mechanisms related to stress and the disruption of maternal homeostasis, or by specific maternally-mediated mechanisms. It is common sense that classification shall not automatically be discounted for substances that produce developmental toxicity only in association with maternal toxicity. A comparison between the severity of the maternal effects and the severity of the findings in the offspring should be performed. The study showed that parental toxicity was confined to changes in body weight gain. The offspring responded partially with inability to survive, small litter size or body weight effects. In the high-dose female population, the dams with total litter loss and /or small litter size (n=6) also revealed body weight gain (BWG) below the group mean value during the gestation period, whereas the remaining females were rather unaffected. Furthermore, the group mean BWG value is in excess of the MTD and is by far exceeded for individual affected animals. According to the Guidance to Regulation (EC) No 1272/2008 on classification, labeling and packaging (CLP), Category 1B has to be selected if “…such data…provide clear evidence of an adverse effect on sexual function and fertility or on development in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of other toxic effects”. On the other hand, classification is not necessary “…in case of minor developmental changes,…small reduction in foetal/pup body weight or retardation of ossification when seen in association with maternal toxicity. In this study, it seems not convincing enough to entirely call offspring toxicity a secondary effect. Furthermore, the extent of offspring toxicity is of a severity degree that cannot be attributed to the rather slight effects seen in the parental animals, only. In the present case, none of the above two options can be applied with enough confidence. Therefore, selection of Category 2 seems to be the most appropriate approach. Therefore, it is proposed to classify the substance for developmental toxicity: Repro category 3 / R63 under Directive 67/548/EEC, as amended for the 28th time in Directive 2001/59/EC and Repro category 2 H361d under CLP Regulation (EC) No. 1272/2008 as amended for the fifth time in EC944/2013.

Additional information