Registration Dossier

Diss Factsheets

Administrative data

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:
21 November 2012 to 12 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD guidance document 43.
Deviations:
no
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
- Strain: Crl:WI rats.
- Age at study initiation: (P) Young adult rats, approximately 10 weeks old at starting of dosing and 12 weeks at mating.
- Weight at study initiation: (P) 362-401 g males and 260-291 g females (did not exceed ± 20% of the mean weight for each sex at onset of treatment).
- Housing: Rats were housed in groups of 5/sex/cage, in Type II and III polypropylene/polycarbonate cages. However during mating and gestation/delivery period, they were housed in pairs or individually, respectively.
- Diet: Complete breeding and maintenance diet for rats and mice, provided ad libitum.
- Water: tap water from municipal supply, provided ad libitum.
- Acclimation period: 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 - 24.9 °C
- Humidity (%): 31 – 70 %
- Air changes (per hr): 15-20 air exchanges/hour.
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(distilled)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
- Method: The test material was formulated in the vehicle at the appropriate concentrations according to the dose level and volume selected. Formulations were prepared according to stability results, within 1 day at room temperature or 7 days when stored refrigerated at 2-8 °C.
- Dosing volume: 20 mL/kg bw/day, the actual volume administered was calculated and adjusted based on each animal’s most recent body weight.
Details on mating procedure:
- Initiation of mating: Mating began 2 weeks after the initiation of treatment.
- M/F ratio per cage: 1:1.
- Length of cohabitation: Females remained with the same male until copulation occurred, or up to 14 days.
- Proof of pregnancy: A vaginal smear was prepared daily for each female during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (considered as Day 0 of pregnancy). There was no female during the study that showed no-evidence of copulation (nonmated).
- After successful mating each pregnant female was caged: Sperm positive females were caged individually. Mating pairs were clearly identified in the data, mating of siblings was avoided.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test formulations for concentration and homogeneity was performed on two occasions during the study. Top, middle and bottom duplicate samples were taken from formulations, during the first and last week of treatment, one set to analyse, collected in replicates as practical, and one set as aback-up, which was not required for confirmatory analyses. Similarly, one sample was taken in duplicate from the Group 1 (control) solution for concentration measurements.

IDENTIFICATION AND QUANTIFICATION OF TEST SUBSTANCE/PRODUCT
- Separation method: HPLC-UV method.
Duration of treatment / exposure:
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post- mating). Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy, 4 days post-partum.
Frequency of treatment:
Daily, on a 7 days/week basis.
Remarks:
Doses / Concentrations:
0.0, 62.5, 250 and 1000 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0.0, 3.125, 12.5 and 50 mg/mL
Basis:
nominal conc.
No. of animals per sex per dose:
12 per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the sponsor in consultation with the Study Director, based on available data and information from previous experimental work, including the results of a 7-day preliminary dose range finding study.
Dose levels were selected with the aim of inducing toxic effects but, if possible, no death or suffering at the highest dose, and a NOAEL.
The oral route was selected as it is a possible route of exposure to the test material in humans.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Signs of morbidity and mortality were recorded twice daily (at the beginning and end of each day). General clinical observations were made once a day, after treatment at approximately the same time each day.
- Cage side observations: Behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were monitored.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Examinations were made once before the first exposure (to allow for within-subject comparisons), and at least once a week thereafter.
- These observations were made outside the home cage in a standard arena, approximately at similar times. Signs evaluated included monitoring of any changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards). Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
On gestation day GD13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).
Females were allowed to litter and rear their offspring and the following observations were recorded: the delivery process, nursing instinct and suckling efficiency. Furthermore, the duration of gestation was recorded and was calculated from day 0 of pregnancy. Dams were observed for nesting behaviour (whether they made a nest from the bedding material and cover their new-borns or not).

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed on Day 0, and at least weekly thereafter until termination. Parent females were weighed on gestation Days GD0, 7, 14 and 20 and on post-partum Days PPD0 (within 24 hours after parturition) and 4 (before termination). The females were additionally weighed on GD3, 10 and 17 in order to give accurate treatment volumes.

FOOD CONSUMPTION: Yes, measured at weekly intervals.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight and epididymis weight.
Special attention during histopathology was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in all F1 offspring, as soon as possible after delivery:
The number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities. Live pups were counted, sexed, weighed individually within 24 hours of parturition (on the first day after parturition was complete, i.e. Day 0 or 1 post-partum) and on Day 4 post-partum with an accuracy of 0.01g.

All the litters were checked and recorded daily for the number of viable and dead pups. All of the pups that were found dead were cannibalised, thus, they were counted and sex determined but were not further examined macroscopically.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were euthanized after 28 days of dosing.
- Maternal animals: All surviving animals were euthanized 4 days post-partum. Females which not delivered were sacrificed 26 days after the last day of mating, as practical.
- Method: Animals were sacrificed under pentobarbital anaesthesia by exsanguination.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cranium, thoracic, the abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues indicated below were weighed, paired organs were weighed individually; absolute organ weights were measured and are reported. Relative organ weights (to body and brain weight) were calculated and are reported.
Uterus (with and without cervix), vagina, testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, ovaries, pituitary.

- Detailed histological examination was performed on the selected list of retained organs in the control and high dose groups, including the brain, pituitary, testis, epididymis, prostate, seminal vesicle with coagulating gland, uterine cervix+body+horn, ovary, vagina and all macroscopic findings (abnormalities) from all animals. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Postmortem examinations (offspring):
SACRIFICE
- Pups euthanized at post-partum day 4.
- These animals were carefully examined at least externally for gross abnormalities.
Statistics:
A statistical evaluation was performed with a statistical program package. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Reproductive indices:
Male Mating Index
Female Mating Index
Male Fertility Index
Female Fertility Index
Gestation Index
Offspring viability indices:
Survival Index
Post-natal mortality
Pre-implantation mortality
Intrauterine mortality
Total mortality
Sex ratio
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related findings.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment related findings.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment related findings.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related findings.
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No treatment related findings.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No treatment related findings.
Reproductive performance:
no effects observed
Description (incidence and severity):
No treatment related findings.
CLINICAL SIGNS AND MORTALITY
There was no unscheduled mortality during this study.
No test material related clinical signs were noted during the study, either at the daily clinical observation or at the weekly evaluation.
In one female treated at 1000 mg/kg bw/day (4509, high-dose group) a subcutaneous mass in the urogenital region was observed from Day 30. This observation was regarded as an incidental finding, unrelated to treatment.

BODY WEIGHT AND FOOD CONSUMPTION
No adverse effects on body weight or body weight gain were observed.
Body weights were comparable to the control in all treated groups. Compared to the control mean, slightly higher body weight gain values were recorded in the treated females during the mating period (Day 14-G0; p<0.05 or p<0.01). These differences were regarded as normal biological variation, not associated with the treatment.
When compared to the control, there were no statistically or toxicologically significant differences in food consumption in the male and female animals administered with the test material up to and including 1000 mg/kg bw/day.

REPRODUCTIVE FUNCTION: SPERM MEASURES: see “histopathology”.

REPRODUCTIVE PERFORMANCE
There were no differences between the control and treated groups with regard to reproductive ability or in the mating, fertility and gestation indices, which showed minor variations within the normal biological ranges, unrelated to treatment.
The mating indices were 100 % in all groups, while fertility indices were 92 % in the control (1/12 non-pregnant female), 100 % in the low- and mid-dose groups and 83 % in the high-dose animals (2/12 non-pregnant females, variation considered incidental).
The gestation index was 100 % in all the dose groups. There were no differences between the control and treated groups with regard to reproductive ability or in the mating, fertility and gestation indices, which showed minor variations within the normal biological ranges, unrelated to treatment.
The mating indices were 100 % in all groups, while fertility indices were 92 % in the control (1/12 non-pregnant female), 100 % in the low- and mid-dose groups and 83 % in the high-dose animals (2/12 non-pregnant females, variation considered incidental).
The gestation index was 100% in all the dose groups.
The administration of the test material was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within up to 5 days of pairing (cohabitation), with the exception of the low-dose female 2503 and the high-dose female 4505, which had a sperm positive vaginal smear after 14 or 13 days of mating, respectively. These variations were regarded as individual, normal biological variability, without toxicological significance.
There was no effect of treatment noted during gestation, parturition or the post-partum period.
The mean duration of pregnancy was similar in the control and treated groups and varied between 21 and 23 days.
All the parturitions were normal.
The numbers of corpora lutea and implantation sites were similar in the treated groups up to and including 1000 mg/kg bw/day, compared to the controls. There were no treatment related adverse effects on the pre/post-implantation, post-natal or total mortality values (%) in any of the dose groups.

ORGAN WEIGHTS
There were no statistically significant differences or changes considered to reflect an adverse effect on organ weights in either males or females in any of the dose groups evaluated, up to and including 1000 mg/kg bw/day.
Statistically significant variations were noted in the absolute and/or relative organ weights (e.g. decreased absolute mean right testes weight in the low- and mid-dose males, or decreased absolute and relative to the brain weight of the vagina in the mid dose females) however, there was no consistent dose response or there were no correlated pathological and histopathological findings, thus, these variations were regarded as normal variations unrelated to treatment.

GROSS PATHOLOGY
No treatment related gross changes were observed at dose levels up to 1000 mg/kg bw/day.
Enlargement of the right or left testis in 1/12 mid- and 1/12 high-dose males, uterine dilatation in 1/12 control female, subcutaneous mass/firm (brown at the cut surface, 2x1x0.5 cm in size) at the skin/subcutis within the mammary gland area in 1/12 high dose female and two placentas present in left uterine horn 1/12 low-dose female were observed at necropsy.

HISTOPATHOLOGY
No treatment related microscopic findings were observed in the experimental animals.
Histopathological evaluation of the male gonads as well as testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoa were similar in the control and high-dose males. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were of similar histological structure in both the control and high-dose females.
There was no meaningful difference between incidence and severity of microscopic changes in the reproductive organs in the high-dose males compared to the control males. The incidence and severity overall were low and without any relationship to treatment and considered to be in the range of common background. Minimal focal unilateral tubular degeneration/atrophy was observed in a control male. Mild sperm reduction of the right epididymis and mild tubular dilatation of the right testis in one high-dose male were seen. Mild tubular dilation was also recorded in one mid-dose (No. 3003) and one high-dose males (No. 4004). In the prostate, minimal mixed mononuclear infiltrate was present in a control male.
Additionally, there was an incidental occurrence of mammary gland hyperplasia (lobular with focal atypia) in one high-dose female (No. 4509). This microscopic lesion correlated with gross change at the skin/subcutis within the mammary gland area in this female. There were no histopathological changes in reproductive organs including the ovary, uterus and vagina in this female rat.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at the maximum dose tested.
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related findings.
Mortality / viability:
no mortality observed
Description (incidence and severity):
No treatment related findings.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment related findings.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related findings.
Histopathological findings:
not examined
VIABILITY AND CLINICAL SIGNS
There were no adverse effects on pups’ viability or any clinical observations recorded in the F1 generation that could be considered toxicologically relevant, or related to test material administration. The mean and total number of viable pups as well as pups survival indices showed no statistically or toxicologically significant differences to control.
There were no pups found dead and intact. A low number of pups were found cannibalized, 2/150 (Day 2 or day 4), 3/176 (Day 0 or Day 1), 2/182 (Day 2) and 1/145 (Day 2) in the control, low-, mid- and high-dose groups, respectively, and 1/182 mid-dose pup was noted to be gray on Day 0; these minor variations were regarded as incidental and ascribed to normal biological variability, unrelated to treatment.

BODY WEIGHT
There was no adverse effect of treatment on the offspring body weight or body weight gain.
Mean body weight and body weight gain values evaluated for all pups or on a litter basis were similar in all treated groups.

INDICES
The sex ratios were similar in the control and treated groups.

GROSS PATHOLOGY
There were no abnormalities at the external examination of all pups surviving to PND4.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at the maximum dose tested.
Reproductive effects observed:
not specified

ANALYSIS OF DOSE FORMULATIONS
No test material was detected in the Control solution samples. All formulations were shown to be homogeneous. All formulations were found to be in the range of 100 to 107 % of nominal concentrations. Based on these results, the formulations were considered suitable for the study purposes. The analytical results are summarized in the Table 1.

Table 1: Dose Formulation Analysis

Sampling Time

Nominal Concentration (mg/mL)

Measured Concentrations ± SD (mg/mL)

Measured Concentration as % of Nominal

First Week

3.125

3.19 ± 0.03

102

12.5

12.5 ± 0.25

100

50

51.5 ± 1.00

103

Last Week

3.125

3.24 ± 0.08

104

12.5

13.4 ± 0.17

107

50

53.1 ± 1.40

106

Table 2: Reproductive Ability Assessment and Indices

 

Dose (mg/kg bw/day)

Control

62.5

250

1000

Paired males – females

12/12

12/12

12/12

12/12

Mated males – females

12/12

12/12

12/12

12/12

Infertile males – Non-pregnant females

1/12

0/12

0/12

2/12

Pregnant females, but not delivered

0/11

0/12

0/12

0/10

Male-female Mating Index (%)

100

100

100

100

Male-female Fertility Index (%)

92

100

100

83

Gestation Index (%)

100

100

100

100

Duration of mating period (days)#

1-4

1-14

1-4

1-13

Surviving females showing evidence of copulation (N)

12/12

12/12

12/12

12/12

Females achieving pregnancy (N)

11/12

12/12

12/12

10/12

# from paring up to successful coitus/sperm positive and/or vaginal plug.

Table 3: Gestation, Parturition and Post-Partum Period

 

Dose (mg/kg bw/day)

Control

62.5

250

1000

Number of treated females

12

12

12

12

Number of surviving treated females

12

12

12

12

Number of dams which delivered/dams with implantations

11/12

12/12

12/12

10/12

Duration of pregnancy (days, mean)

22.27

22.25

22.33

22.10

Number of corpora lutea/dams (mean)

17.27

18.00

18.00

18.10

Number of implantations/dams (mean)

14.55

15.25

15.83

15.20

Number of dams with live pups day 0

11

12

12

10

Number of dams with live pups day 4

11

12

12

10

Pre-implantation mortality

2.73

2.75

2.17

2.90

Intrauterine mortality

0.91

0.75

0.67

0.70

Post-natal mortality

0.18

0.08

0.17

0.10

Total mortality

1.09

0.83

0.83

0.80

Table 4: Offspring Generation

Females

Dose (mg/kg bw/day)

Control

62.5

250

1000

Number of liveborns (total) on PND0

150

174

182

145

Number of viable pups (mean) on PND0

13.64

14.50

15.17

14.50

Number of viable pups (mean) on PND4

13.45

14.42

15.00

14.40

Number of viable pups (total) on PND0

150

174

182

145

Number of viable pups (total) on PND4

148

173

180

144

Survival index PND0

100.00

98.99

100.00

100.00

Survival index PND0-4

98.69

98.23

98.91

99.29

Sex ratio (%) on PND0

47.32

54.45

54.53

56.25

Sex ratio (%) on PND4

46.71

54.22

54.97

56.58

Conclusions:
Under the conditions of the test, no adverse effects were observed as a direct result of treatment up to and including 1000 mg/kg bw/day. Therefore the NOEC for parental animals and reproductive toxicity was determined to be 1000 mg/kg bw/day.
Executive summary:

The roxicity to reproduction, of the test material, was determined in a reproduction/developmental toxicity screening study performed using Wistar rats. The study was preofmed under GLP conditions and in line with the standardised guidelines OECD 421 and OECD guidance document 43.

During the study groups of 12 male and 12 female rats were exposed to the test material, formulated in distilled water, at concentrations of 0.0, 62.5, 250 and 1000 mg/kg bw/day via oral gavage. Animals were dosed daily at 20 mL/kg bw/day, 7 days per week. Males were dosed for 28 days (14 days pre-mating and 14 days mating/post- mating). Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy, 4 days post-partum.

Analytical verification confirmed that all formulations within the range of 100 to 107 % of nominal concentrations, and were thus suitable for the purpose of the study.

Based on the detailed parental observations, no adverse effects were recorded as a direct result of treatment up to and including 1000 mg/kg bw/day. Reproductive indices revealed no test material-related effects on fertility. Offspring indices and post-mortem examination of the pups revealed no treatment related effects.

Therefore the NOAEL for maternal, and reproductive, toxicity was determined to be 1000 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The overall quality of the dataset is considered to be high.
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 the key study (Kiss, 2013), the toxicity of the test material to reproduction was determined in a reproduction/developmental toxicity screening study which was performed using Wistar rats under GLP conditions and in line with standardised guidelines. The study was assigned a reliability score of 1 in accordance with Klimisch (1997). During the study groups of 12 male and 12 female rats were exposed to the test material, formulated in distilled water, at concentrations of 0.0, 62.5, 250 and 1000 mg/kg bw/day via oral gavage. Animals were dosed daily at 20 mL/kg bw/day, 7 days per week. Males were dosed for 28 days (14 days pre-mating and 14 days mating/post- mating). Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy, 4 days post-partum. Analytical verification confirmed that all formulations within the range of 100 to 107 % of nominal concentrations, and were thus suitable for the purpose of the study. Based on the detailed parental observations no adverse effects were recorded as a direct result of treatment up to and including 1000 mg/kg bw/day. Reproductive indices revealed no effects on fertility. Offspring indices and post-mortem examination of the pups revealed no treatment related effects. Therefore the NOAEL for both parental, and reproductive, toxicity was determined to be 1000 mg/kg bw/day.

In accordance with Section 1 of REACH Annex XI, the two-generation reproductive toxicity study (required in section 8.7.3 of Annex IX does not need to be conducted if the study does not appear to be scientifically necessary. No reproductive effects were noted in an OECD 421 screening study, therefore further testing is considered unnecessary.


Short description of key information:
Reproduction NOAEL: 1000 mg/kg bw/day, Parental NOAEL: 1000 mg/kg bw/day, OECD 421, Kiss (2013).

Justification for selection of Effect on fertility via oral route:
The key study was performed in line with the standardised guidelines OECD 421 and OECD guidance document 43, under GLP conditions. The study was performed to a high standard with sufficient reporting to assess the quality of the data. The study was assigned a reliability score of 1 in accordance with the criteria outlined in Klimisch (1997). This study was considered suitable to be the key study and sufficient to address this endpoint.

Effects on developmental toxicity

Description of key information
Developmental NOAEL: 1000 mg/kg bw/day, Maternal NOAEL: 1000 mg/kg bw/day, OECD 421, Kiss (2013).
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 November 2012 to 12 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD guidance document 43.
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Crl:WI rats.
- Age at study initiation: (P) Young adult rats, approximately 10 weeks old at starting of dosing and 12 weeks at mating.
- Weight at study initiation: (P) 362-401 g males and 260-291 g females (did not exceed ± 20% of the mean weight for each sex at onset of treatment).
- Housing: Rats were housed in groups of 5/sex/cage, in Type II and III polypropylene/polycarbonate cages. However during mating and gestation/delivery period, they were housed in pairs or individually, respectively.
- Diet: Complete breeding and maintenance diet for rats and mice, provided ad libitum.
- Water: tap water from municipal supply, provided ad libitum.
- Acclimation period: 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 - 24.9 °C
- Humidity (%): 31 – 70 %
- Air changes (per hr): 15-20 air exchanges/hour.
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(distilled)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
- Method: The test material was formulated in the vehicle at the appropriate concentrations according to the dose level and volume selected. Formulations were prepared according to stability results, within 1 day at room temperature or 7 days when stored refrigerated at 2-8 °C.
- Dosing volume: 20 mL/kg bw/day, the actual volume administered was calculated and adjusted based on each animal’s most recent body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test formulations for concentration and homogeneity was performed on two occasions during the study. Top, middle and bottom duplicate samples were taken from formulations, during the first and last week of treatment, one set to analyse, collected in replicates as practical, and one set as aback-up, which was not required for confirmatory analyses. Similarly, one sample was taken in duplicate from the Group 1 (control) solution for concentration measurements.

IDENTIFICATION AND QUANTIFICATION OF TEST SUBSTANCE/PRODUCT
- Separation method: HPLC-UV method.
Details on mating procedure:
- Initiation of mating: Mating began 2 weeks after the initiation of treatment.
- M/F ratio per cage: 1:1.
- Length of cohabitation: Females remained with the same male until copulation occurred, or up to 14 days.
- Proof of pregnancy: A vaginal smear was prepared daily for each female during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (considered as Day 0 of pregnancy). There was no female during the study that showed no-evidence of copulation (nonmated).
- After successful mating each pregnant female was caged: Sperm positive females were caged individually. Mating pairs were clearly identified in the data, mating of siblings was avoided.
Duration of treatment / exposure:
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post- mating). Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy, 4 days post-partum.
Frequency of treatment:
Daily, on a 7 days/week basis.
No. of animals per sex per dose:
12 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the sponsor in consultation with the Study Director, based on available data and information from previous experimental work, including the results of a 7-day preliminary dose range finding study.
Dose levels were selected with the aim of inducing toxic effects but, if possible, no death or suffering at the highest dose, and a NOAEL.
The oral route was selected as it is a possible route of exposure to the test material in humans.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Signs of morbidity and mortality were recorded twice daily (at the beginning and end of each day). General clinical observations were made once a day, after treatment at approximately the same time each day.
- Cage side observations: Behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were monitored.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Examinations were made once before the first exposure (to allow for within-subject comparisons), and at least once a week thereafter.
- These observations were made outside the home cage in a standard arena, approximately at similar times. Signs evaluated included monitoring of any changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards). Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
On gestation day GD13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).
Females were allowed to litter and rear their offspring and the following observations were recorded: the delivery process, nursing instinct and suckling efficiency.

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed on Day 0, afterwards at least weekly and at termination. Parent females were weighed on gestation Days GD0, 7, 14 and 20 and on post-partum Days PPD0 (within 24 hours after parturition) and 4 (before termination). The females were additionally weighed on GD3, 10 and 17 in order to give accurate treatment volumes.

FOOD CONSUMPTION: Yes, measured at weekly intervals.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day.

SACRIFICE
- Maternal animals: All surviving animals were euthanized 4 days post-partum. Females which not delivered were sacrificed 26 days after the last day of mating, as practical.
- Method: Animals were sacrificed under pentobarbital anaesthesia by exsanguination.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cranium, thoracic, the abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues indicated below were weighed, paired organs were weighed individually; absolute organ weights were measured and are reported. Relative organ weights (to body and brain weight) were calculated and are reported.
Uterus (with and without cervix), vagina, testes, brain, ovaries, pituitary.

- Detailed histological examination was performed on the selected list of retained organs in the control and high dose groups, including the brain, pituitary, uterine cervix+body+horn, ovary, vagina and all macroscopic findings (abnormalities) from all animals. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
Fetal examinations:
- Pups euthanized at post-partum day 4.
- These animals were carefully examined at least externally for gross abnormalities.
Statistics:
The statistical evaluation of appropriate data was performed with a statistical program package. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Indices:
Male Mating Index
Female Mating Index
Male Fertility Index
Female Fertility Index
Gestation Index
Survival Index
Post-natal mortality
Pre-implantation mortality
Intrauterine mortality
Total mortality
Sex ratio
Details on maternal toxic effects:
Maternal toxic effects:no effects. Remark: No treatment related effects observed up to 1000 mg/kg bw/day.

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
There was no unscheduled mortality during this study.
No test material related clinical signs were noted during the study, either at the daily clinical observation or at the weekly evaluation.
In one female treated at 1000 mg/kg bw/day (4509, high-dose group) a subcutaneous mass in the urogenital region was observed from Day 30. This observation was regarded as an incidental finding, unrelated to treatment.

BODY WEIGHT AND FOOD CONSUMPTION
No adverse effects on body weight or body weight gain were observed.
Body weights were comparable to the control in all treated groups. Compared to the control mean, slightly higher body weight gain values were recorded in the treated females during the mating period (Day 14-G0; p<0.05 or p<0.01). These differences were regarded as normal biological variation, not associated with the treatment.
When compared to the control, there were no statistically or toxicologically significant differences in food consumption in the female animals administered with the test material up to and including 1000 mg/kg bw/day.

REPRODUCTIVE PERFORMANCE
There were no differences between the control and treated groups with regard to reproductive ability or in the mating, fertility and gestation indices, which showed minor variations within the normal biological ranges, unrelated to treatment.
The administration of the test material was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within up to 5 days of pairing (cohabitation), with the exception of the low-dose female 2503 and the high-dose female 4505, which had a sperm positive vaginal smear after 14 or 13 days of mating. These variations were regarded as individual, normal biological variability, without toxicological significance.
There was no effect of treatment noted during gestation, parturition or the post-partum period.
The mean duration of pregnancy was similar in the control and treated groups and varied between 21 and 23 days.
All the parturitions were normal.
The numbers of corpora lutea and implantation sites were similar in the treated groups up to and including 1000 mg/kg bw/day, compared to the controls. There were no treatment related adverse effects on the pre/post-implantation, post-natal or total mortality values (%) in any of the dose groups.

ORGAN WEIGHTS
There were no statistically significant differences or changes considered to reflect an adverse effect on organ weights in females at any of the dose groups evaluated, up to and including 1000 mg/kg bw/day.
A Statistically significant decreased in absolute and relative weight was observed in the brain and the vagina in the mid dose females. However, there was no consistent dose response or there were no correlated pathological and histopathological findings, thus, these variations were regarded as normal variations unrelated to treatment.

GROSS PATHOLOGY
No treatment related gross changes were observed at dose levels up to 1000 mg/kg bw/day.
Uterine dilatation in 1/12 control female, subcutaneous mass/firm (brown at the cut surface, 2x1x0.5 cm in size) at the skin/subcutis within the mammary gland area in 1/12 high dose female and two placentas present in left uterine horn 1/12 low-dose female were observed at necropsy.

HISTOPATHOLOGY
No treatment related microscopic findings were observed in the experimental animals.
The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were of similar histological structure in both the control and high-dose females.
There was an incidental occurrence of mammary gland hyperplasia (lobular with focal atypia) in one high-dose female (No. 4509). This microscopic lesion correlated with gross change at the skin/subcutis within the mammary gland area in this female. There were no histopathological changes in reproductive organs including the ovary, uterus and vagina in this female rat.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: No treatment related effects observed up to 1000 mg/kg bw/day.

Details on embryotoxic / teratogenic effects:
VIABILITY AND CLINICAL SIGNS
There were no adverse effects on pups’ viability or any clinical observations recorded in the F1 generation that could be considered toxicologically relevant, or related to test material administration. The mean and total number of viable pups as well as pups survival indices showed no statistically or toxicologically significant differences to control.
There were no pups found dead and intact. A low number of pups were found cannibalized, 2/150 (Day 2 or day 4), 3/176 (Day 0 or Day 1), 2/182 (Day 2) and 1/145 (Day 2) in the control, low-, mid- and high-dose groups, respectively, and 1/182 mid-dose pup was noted to be gray on Day 0; these minor variations were regarded as incidental and ascribed to normal biological variability, unrelated to treatment.

BODY WEIGHT
There was no adverse effect of treatment on the offspring body weight or body weight gain.
Mean body weight and body weight gain values evaluated for all pups or on a litter basis were similar in all treated groups.

INDICES
The sex ratios were similar in the control and treated groups.

GROSS PATHOLOGY
There were no abnormalities at the external examination of all pups surviving to PND4.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at the maximum dose tested.
Abnormalities:
not specified
Developmental effects observed:
not specified

ANALYSIS OF DOSE FORMULATIONS
No test material was detected in the Control solution samples. All formulations were shown to be homogeneous. All formulations were found to be in the range of 100 to 107 % of nominal concentrations. Based on these results, the formulations were considered suitable for the study purposes. The analytical results are summarized in the Table 1.

Table 1: Dose Formulation Analysis

Sampling Time

Nominal Concentration (mg/mL)

Measured Concentrations ± SD (mg/mL)

Measured Concentration as % of Nominal

First Week

3.125

3.19 ± 0.03

102

12.5

12.5 ± 0.25

100

50

51.5 ± 1.00

103

Last Week

3.125

3.24 ± 0.08

104

12.5

13.4 ± 0.17

107

50

53.1 ± 1.40

106

Table 2: Offspring Generation

Females

Dose (mg/kg bw/day)

Control

62.5

250

1000

Number of liveborns (total) on PND0

150

174

182

145

Number of viable pups (mean) on PND0

13.64

14.50

15.17

14.50

Number of viable pups (mean) on PND4

13.45

14.42

15.00

14.40

Number of viable pups (total) on PND0

150

174

182

145

Number of viable pups (total) on PND4

148

173

180

144

Survival index PND0

100.00

98.99

100.00

100.00

Survival index PND0-4

98.69

98.23

98.91

99.29

Sex ratio (%) on PND0

47.32

54.45

54.53

56.25

Sex ratio (%) on PND4

46.71

54.22

54.97

56.58

Conclusions:
Under the conditions of the test, no adverse effects were observed as a direct result of treatment up to and including 1000 mg/kg bw/day. Therefore the NOAEL for maternal, and developmental, toxicity was determined to be 1000 mg/kg bw/day.
Executive summary:

The developmental toxicity of the test material was determined in a reproduction/developmental toxicity screening study performed using Wistar rats. The study was conducted under GLP conditions and in line with the standardised guidelines OECD 421 and OECD guidance document 43.

During the study groups of 12 male and 12 female rats were exposed to the test material, formulated in distilled water, at concentrations of 0.0, 62.5, 250 and 1000 mg/kg bw/day via oral gavage. Animals were dosed daily at 20 mL/kg bw/day, 7 days per week. Males were dosed for 28 days (14 days pre-mating and 14 days mating/post- mating). Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy, 4 days post-partum.

Analytical verification confirmed that all formulations within the range of 100 to 107 % of nominal concentrations, and were thus suitable for the purpose of the study.

Based on the detailed maternal observations no adverse effects were recorded as a direct result of treatment up to and including 1000 mg/kg bw/day. Offspring indices and post-mortem examination of the pups revealed no treatment related effects.

Therefore the NOAEL for maternal, and developmental, toxicity was determined to be 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The overall quality of the dataset is considered to be high.
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 the key study (Kiss, 2013), the developmental toxicity of the test material was determined in a reproduction/developmental toxicity screening study performed using Wistar rats under GLP conditions and in line with the standardised guidelines. The study was assigned a reliability score of 1 in line with Klimisch (1997).

During the study groups of 12 male and 12 female rats were exposed to the test material, formulated in distilled water, at concentrations of 0.0, 62.5, 250 and 1000 mg/kg bw/day via oral gavage. Animals were dosed daily at 20 mL/kg bw/day, 7 days per week. Males were dosed for 28 days (14 days pre-mating and 14 days mating/post- mating). Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy, 4 days post-partum.

Analytical verification confirmed that all formulations within the range of 100 to 107 % of nominal concentrations, and were thus suitable for the purpose of the study.

Based on the detailed maternal observations no adverse effects were recorded as a direct result of treatment up to and including 1000 mg/kg bw/day. Offspring indices and post-mortem examination of the pups revealed no treatment related effects.

Therefore the NOAEL for maternal, and developmental, toxicity was determined to be 1000 mg/kg bw/day.

In accordance with Section 1 of Annex XI, the pre-natal developmental toxicity study, as required in Section 8.7.2 of Annex IX, does not appear scientifically necessary. No developmental effects were noted in an OECD 421 screening study, therefore further testing is considered unnecessary.


Justification for selection of Effect on developmental toxicity: via oral route:
The key study was performed in line with the standardised guidelines OECD 421 and OECD guidance document 43, under GLP conditions. The study was performed to a high standard with sufficient reporting to assess the quality of the data. The study was assigned a reliability score of 1 in accordance with the criteria outlined in Klimisch (1997). This study was considered suitable to be the key study and sufficient to address this endpoint.

Justification for classification or non-classification

In accordance with criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the test material does not require classification for reproductive or developmental toxicity.

Additional information