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Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 July 2012 to 27 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to an internationally recognized protocol and all relevant GLP requirements were fulfilled.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
The study integrity was not adversely affected by the deviations.
Qualifier:
equivalent or similar to
Guideline:
other: US EPA OPPTS 870.3650, July 2000
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: CrI:WI(Han) (outbred, SPF-Quality)
Sex:
male/female
Details on test animals and environmental conditions:
Refer to Section 7.5.1
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Refer to Section 7.5.1
Details on mating procedure:
Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).

Post-mating Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males. Detection of mating was not confirmed for animal nos. 43 (Group 1), 58 (Group 2) and 62 (Group 3) which did deliver live offspring. The mating date of these animals were estimated at 21 days prior to the actual delivery dates. These days were designated Day 0 post-coitum.

The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Refer to Section 7.5.1
Duration of treatment / exposure:
After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg/day. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 43 - 53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.
Frequency of treatment:
Refer to Section 7.5.1
Details on study schedule:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 43-53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 41, 43 (Group 1), 62 , 70 (Group 3) and 77 (group 4) were not dosed during littering.

Remarks:
Doses / Concentrations:
0 (vehicle only), 100, 300 and 1000 mg/kg/d
Basis:
nominal in water
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
PREPARATION OF DOSING SOLUTIONS:

Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No correction was made for the purity/composition of the test substance.

Doses were administered by plastic feeding tube.

Dose volume: 5 mL/kg
Positive control:
None
Parental animals: Observations and examinations:
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Oestrous cyclicity (parental animals):
Not determined
Sperm parameters (parental animals):
Parameters examined in selected 5 male parental animals:

testis weight, epididymis weight, prostate weight (after at least 24 h fixation), and weight of seminal vesicles including the coagulating glands

Refer to Section 7.5.1 for all histopathological analyses conducted in this study.
Litter observations:
Each litter was examined for the following if practicle and possible:

Mortality / Viability
The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.

Clinical signs

At least once daily, detailed clinical observations were made for all animals.

Body weights
Live pups were weighed on Days 1 and 4 of lactation.

Sex
Sex was determined for all pups on Days 1 and 4 of lactation.
Postmortem examinations (parental animals):
Refer to Section 7.5.1 of this IUCLID file
Postmortem examinations (offspring):
Necropsy

Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.
Statistics:
Refer to Section 7.5.1 of this IUCLID file
Reproductive indices:
Mating index (%): No. females mated / No. females paired x 100
Fertility index (%): No. pregnant females / No. of females placed x 100
Conception index (%): No. pregnant females / No. females mated x 100
Gestation index (%): No. females bearing live pups / No. pregnant females x 100
Duration ofgestation: No. days between confirmation of mating and the beginning of parturition.
Offspring viability indices:
Percentage live males at first litter check.
Percentage live females at first litter check.
Percentage of postnatal loss.
Viability index (%): No. live pups before necropsy / No. of pups born alive x 100
Clinical signs:
no effects observed
Description (incidence and severity):
Refer to Section 7.5.1 of this IUCLID file
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Refer to Section 7.5.1 of this IUCLID file
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Refer to Section 7.5.1 of this IUCLID file
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Refer to Section 7.5.1 of this IUCLID file
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Refer to Section 7.5.1 of this IUCLID file for a more complete description of results.

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg). No treatment-related toxicologically relevant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on a lack of significant reproductive effects measured in the study.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
At 1000 mg/kg, sex ratio of the pups indicated more female than male pups (62% female pups compared to 47% of the control group); this was statistically significantly different from controls.

No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).
Reproductive effects observed:
not specified

Reproduction data summary

   Control  100 mg/kg  300 mg/kg  1000 mg/kg
 Females paired  10  10  10  10
 Females mated  10  9  10  9
 Females non-mated  0  1  0  1
 Pregnant females  9  9  10  9
 Non-pregnant females  1  0  0  0
 Females with living pups on Day 1  9  9  10  9
 Mating index (%)  100.0  90.0  100.0  90.0
 Fertility index (%)  90.0  90.0   100.0  90.0
 Conception index (%)  90.0  100.0   100.0  100.0
Gestation index (%)   100.0  100.0   100.0  100.0
Conclusions:
No significant reproductive toxicity was noted up to a dose of 1000 mg/kg/d. The NOAEL for reproductive toxicity is 1000 mg/kg.
Executive summary:

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg). No treatment-related toxicologically relevant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).

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
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 an OECD 422 guideline study, the subject material was administered daily by gavage to groups of 10 each male and female rats at dose levels of 0 (control), 100, 300 or 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). Females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and for at least 4 days of lactation (43 -53 days).

 

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg). No treatment-related toxicologically relevant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).


Short description of key information:
An OECD 422 test (combined repeated dose toxicity with reproductive / developmental toxicity) conducted by oral gavage was conducted on the subject material.

Justification for selection of Effect on fertility via oral route:
Lack of significant reproductive effects were reported in a guideline study (OECD 422).

Effects on developmental toxicity

Description of key information
An OECD 422 test (combined repeated dose toxicity with reproductive / developmental toxicity) conducted by oral gavage was conducted on the subject material.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 July 2012 to 27 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to an internationally recognized protocol and all relevant GLP requirements were fulfilled.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
other: OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test
Deviations:
yes
Remarks:
The study integrity was not adversely affected by the deviations.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: CrI:WI(Han) (outbred, SPF-Quality)
Details on test animals and environmental conditions:
Refer to Section 7.5.1 of this IUCLID file
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Refer to Section 7.5.1 of this IUCLID file
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Refer to Section 7.5.1 of this IUCLID file
Details on mating procedure:
Refer to Section 7.5.1 and 7.8.1 of this IUCLID file
Duration of treatment / exposure:
Refer to Section 7.5.1 and 7.8.1 of this IUCLID file
Frequency of treatment:
Refer to Section 7.5.1 and 7.8.1 of this IUCLID file
Duration of test:
Refer to Section 7.5.1 and 7.8.1 of this IUCLID file
Remarks:
Doses / Concentrations:
0 (vehicle only), 100, 300 and 1000 mg/kg/d
Basis:
nominal in water
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Refer to Section 7.5.1 and 7.8.1 of this IUCLID file
Maternal examinations:
Refer to Section 7.5.1 and 7.8.1 of this IUCLID file
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
Not applicable
Statistics:
Refer to Section 7.5.1 and 7.8.1 of this IUCLID file
Indices:
Liters (total)
Duration of gestation
Dead pups at first litter check
Living pups at first litter check
Postnatal loss
Viability index (%)
Details on maternal toxic effects:
Maternal toxic effects:no effects. Remark: Refer to Section 7.5.1 of this IUCLID file
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
At 1000 mg/kg, sex ratio of the pups indicated more female than male pups (62% female pups compared to 47% of the control group); this was statistically significantly different from controls. The laboratory’s historical control data (97 screening studies, years 2008-2012) includes two studies in
which a comparable percentage of female pups was noted. As these two historical studies show the ultimate limit of the historical control data range, a possible relationship to treatment could not be excluded for the changed sex ratio in the current study. This was strengthened by the fact that seven out of nine litters in this group showed more female than male pups.

No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).
Abnormalities:
not specified
Developmental effects observed:
not specified

Developmental data summary

   Control  100 mg/kg  300 mg/kg  1000 mg/kg
 Litters        
 Total  9  9  10  9
 Dration of gestation        
 Mean  21.3  21.1  21.3  21.3
 St Dev  0.5  0.6  0.5  0.5
 N  9  9  10  9
 Dead pups at first litter check        
 Litters affected (%)  0  0  0
 Total 1  0  0  0
 Mean  0.1  0  0  0
 St Dev  0.3  0.0  0.0  0.0
 N  9  9  10  9
 Living pups at first litter check        
 % males/%females 53/47   53/47  54/46  38/62#
 Total 93  104   92  102
 Mean 10.3   11.6  9.2  11.3
 St Dev  2.5  3.2  3.7  2.9
 N  9  9  10  9
 Postnatal loss        
 % of living pups  2.2  0.0  1.1  0.0
 Litters affected (No.)  0  1  0
 Total (No.)  2  0  1  0
 Mean 0.2   0.0  0.1  0.0
 St Dev 0.7   0.0  0.3  0.0
 N  9  10  9
 Viability Index (No.)  97.8  100.0  98.9  100.0

# Fischer's exact test significance at 5%

Conclusions:
Developmental toxicity was observed at 1000 mg/kg as a possible relationship to treatment could not be excluded for the change in sex ratio. The NOAEL for developmental effects was 300 mg/kg.
Executive summary:

At 1000 mg/kg, sex ratio of the pups indicated more female than male pups (62% female pups compared to 47% of the control group); this was statistically significantly different from controls. The laboratory’s historical control data (97 screening studies, years 2008-2012) includes two studies in which a comparable percentage of female pups was noted. As these two historical studies show the ultimate limit of the historical control data range, a possible relationship to treatment could not be excluded for the changed sex ratio in the current study. This was strengthened by the fact that seven out of nine litters in this group showed more female than male pups.

It should be noted that this change in sex ratio may be indicative of a decrease in anogenital distance for the male pups leading to a ‘misclassification’ into female pups, rather than a direct effect on sex ratio.

No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
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 an OECD 422 guideline study, the subject material was administered daily by gavage to groups of 10 each male and female rats at dose levels of 0 (control), 100, 300 or 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). Females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and for at least 4 days of lactation (43 -53 days).

 

At 1000 mg/kg, the sex ratio of the pups indicated more female than male pups (62% female pups compared to 47% of the control group); this was statistically significantly different from controls. The laboratory’s historical control data (97 screening studies, years 2008-2012) includes two studies in which a comparable percentage of female pups was noted. As these two historical studies show the ultimate limit of the historical control data range, a possible relationship to treatment could not be excluded for the changed sex ratio in the current study. This was strengthened by the fact that seven out of nine litters in this group showed more female than male pups.

It should be noted that this change in sex ratio may be indicative of a decrease in anogenital distance for the male pups leading to a ‘misclassification’ into female pups, rather than a direct effect on the sex ratio.

No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).


Justification for selection of Effect on developmental toxicity: via oral route:
Significant developmental effects were reported in a guideline study (OECD 422).

Justification for classification or non-classification

Based on a lack of significant reproductive effects noted in an OECD 422 study in rats, no classification of the test material for reproductive effects is warranted.

Based on significant developmental effects noted in an OECD 422 study in rats, the subject material receives a classification under the EU Dangerous Substance Directive (67/548/EEC) of Repr. Cat. 3; R63 (Possible risk of harm to the unborn child). Classification under the EU Regulation 1272/2008 results in a Repr. Cat. 2 (H361: Suspected of damaging fertility or the unborn child).