Registration Dossier

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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Toxicity to reproduction (OECD 421, Reproduction/Developmental Toxicity Screening Test), rat (m, f): NOAEL systemic = 60 mg/kg bw/day, NOAEL reproductive fertility = 60 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-10-02 to 2014-08-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 27 Jul 1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)
Version / remarks:
adopted Jul 2000
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No. 440/2008, L 142, Appendix Part B
Version / remarks:
adopted 30 May 2008
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, 97633 Sulzfeld, Germany
- Age at study initiation: 10-11 weeks (males) and 10-11 weeks (females)
- Weight at study initiation: 270 - 318 g (males) and 184 - 219 g (females)
- Housing: individually in IVC cages (except during the mating period when one female was paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was ground to a fine powder and was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulation was mixed thoroughly using a homogenizer. The test item formulation was prepared once a week and stored at ambient temperature. Every day 20 minutes before the dose administration the formulation was kept on a magnetic stirrer to ensure the homogeneity. Homogeneity of the test item in the vehicle was also maintained by keeping the prepared suspension on a magnetic stirrer during dose administration.

VEHICLE
- Concentration in vehicle: 3, 6 and 12 mg/mL
- Amount of vehicle: 5 mL/kg bw/day
- Lot/batch no.: 1101401034
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until mating was confirmed
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged: individually in IVC cages type III H polysulphone cages on Altromin saw fibre bedding
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration analysis of formulation samples was determined in study week 1, 2, 4 and 7 for all dose groups. The mean recoveries observed at 15, 30 and 60 mg/kg bw/day were 105.4%, 97.0% and 97.5% of the nominal concentration, respectively.
Stability of formulation samples was investigated for the 15 and 60 mg/kg bw/day dose groups. After storage at room temperature for 7 days recovery compared to starting value was between 93.6% and 106.7%.
Homogeneity of formulation samples was determined in study week 1 and 7 for formulations of the dose groups 15 and 60 mg/kg bw/day. The mean recovery observed for the 15 mg/kg bw/day dose group was between 91.9% and 93.0% of the nominal value and between 90.4% and 94.5% of the nominal value for the 60 mg/kg bw/day dose group. The coefficients of variation of the different sampling locations (top, middle and bottom) were between 1.3% and 2.4% at 15 mg/kg bw/day and between 1.2% and 1.7% at 60 mg/kg bw/day.
Duration of treatment / exposure:
The females were treated with the test item formulation or vehicle 7 days per week for a period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating, during the gestation period and up to post-natal day 3.
Males were dosed during 14 days of pre-mating and maximum 14 days of mating. After the mating period until the minimum total dosing period of 28 days were completed.
Frequency of treatment:
daily, 7 days/week
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected on the basis of data from a dose range-finding study, and a 28-day study started 3 weeks prior to this study in which mortality was observed at the dose levels 125 and 100 mg/kg bw/day.

Parental animals: Observations and examinations:
OBSERVATIONS: Yes
- Time schedule: The animals were checked once daily for clinical signs and twice daily for morbidity and mortality, except for weekends and public holidays when observations were made once daily.
- Observations included: Spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. In addition, changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups. The animal prematurely sacrificed was weighed prior to the sacrifice.

FOOD CONSUMPTION: Yes
- Time schedule for food consumption: Food consumption was measured weekly on the same days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

FOOD EFFICIENCY: No
Sperm parameters (parental animals):
Parameters examined in male parental generation P0: testis weight and epididymis weight.
For the testes a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS stained slides.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in the F1 offspring: number and sex of pups, stillbirths, live births, runts and the presence of gross anomalies. Live pups were counted and sexed and litters weighed within 24 h of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by the tattoo mark on the paw. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.

GROSS EXAMINATION OF DEAD PUPS: Yes, the dead pups and pups sacrificed on day 4 post partum were carefully examined externally for gross abnormalities.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study Day 29.
- Maternal animals: Females which delivered were sacrificed on post-natal day 4 (PND 4).
- Non-pregnant females were sacrificed on study Day 26 from the day of evidence of mating.

GROSS NECROPSY
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for each parental female. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved in 10% neutral buffered formalin, except for testes and epididymides which were fixed in modified Davidson’s Solution for 24 hours and transferred to 70% ethanol.

ORGAN WEIGHTS
The following organs were weighed for all selected animals at necropsy: brain, stomach, small and large intestines (including Peyer's patches), liver, thymus, spleen, ovaries, uterus with cervix, lung, urinary bladder, lymph nodes (mesenteric and axillary), trachea, kidneys, adrenal glands, heart, testes, epididymides and all gross lesions.

HISTOPATHOLOGY
Tissues / Organs examined by light microscopy were: epididymides, ovaries, prostate gland, seminal vesicles with coagulating glands, testes, uterus with cervix, vagina and all gross lesions.


Postmortem examinations (offspring):
SACRIFICE:
- All surviving pups were sacrificed by decapitation on PND 4.

GROSS NECROPSY:
- Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.

HISTOPATHOLOGY / ORGAN WEIGHTS: not performed
Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).
Reproductive indices:
Copulation Index (%) = (No. of rats copulated / No. of pairs) X 100%
Fertility Index (%) = (No. of females pregnant / No. of females copulated) X 100%
Delivery Index (%) = (No. of dams with live new borns / No. of pregnant dams) X 100%
Offspring viability indices:
Viability Index (%) = (No. of live offspring at day 4 / No. of live offspring at birth) X 100%
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs that were considered to be an adverse effect of treatment. Clinical signs were observed in all treatment and control groups and comprised slight to moderate and/ or severe diarrhea, piloerection, salivation and/ or nasal discharge in both sexes. The incidences of diarrhea and salivation in males and nasal discharge and salivation in females were similar in control animals. However, the incidences of moving the bedding, abnormal breathing, piloerection and nasal discharge in males, and moving the bedding, abnormal breathing and diarrhea in females were observed at a higher frequency in test item-treated animals, with an increased severity at higher doses. Abnormal breathing, piloerection and nasal discharge in males and/ or females were transient. The observations were made more frequently in the second half of the study (after mating) and considered to be treatment-related. Incidences of moving the bedding and salivation were considered to be due to discomfort caused to the animals during the test item treatment. Other findings such as alopecia, eschar and slightly discolored green feces in single isolated animals at 30 and 60 mg/kg bw/day were considered to be incidental. For details please refer to Table no. 1 under "Any other information on results incl. tables".
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
30 mg/kg bw/day: 1/10 females died on gestation day 5, treatment-related, the cause was considered to be accidental influx into the respiratory tract of the test substance but not due to systemic toxicity.
60 mg/kg bw/day: 1/10 females was killed for ethical reasons on PND 2, treatment-related, the cause was considered to be accidental influx into the respiratory tract of the test substance but not due to systemic toxicity.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
30 mg/kg bw/day: Statistically significant decrease in body weight gain in females in the first week of gestation, transient finding, non-adverse.
60 mg/kg bw/day: Decreased mean body weight in males after 2 weeks of treatment (-7 to -5% of control), statistically significant from week 3 onwards, treatment-related, considered to be a result of local irritative effects of the test item in the forestomach.
(For details please refer to Table no. 2 under "Any other information on results incl. tables").
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
60 mg/kg bw/day: statistically significantly decreased in males during the pre-mating period, in line with reduced body weight gain, considered to be a result of local irritative effects of the test item in the forestomach; slightly (statistically not significantly) decreased in females after 2 weeks of treatment, during the last week of gestation and during the lactation period, observations in females were within the normal biological variation.
(For details please refer to Table no. 3 under "Any other information on results incl. tables").
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were no test item-related abnormalities noted in surviving animals. 1/10 females at 30 mg/kg bw/day and 1/10 females at 60 mg/kg bw/day that died during the treatment period or were killed in extremis showed necrotising inflammation of the trachea or nose and findings in the lungs (flocculent and fluid contents in the bronchi, bronchiole and alveolar space and tracheal mucosal epithelial hypertrophy and alveolitis at/around broncheolar/alveolar duct area of the lung) which indicated an accidental regurgitation/aspiration of the test item. Both females had histopathological findings in the forestomach. Epithelial squamous hyperplasia, hyperkeratosis, parakeratosis were noted in both animals, whereas submucosal edema and dyskeratosis were recorded only in the dead female of the high dose group. The findings were attributed to local irritation caused by the test substance but not to systemic toxicity. Both animals showed thymic atrophy and/or splenic lymphoid atrophy and adrenocortical diffuse hypertrophy, which were related to stress. Sertoli cell vacuolisation and/or spermatid retention were recorded at minimal severity in 4/10, 0/10, 1/10 and 2/10 and in 1/10, 0/10, 1/10 and 0/10 males at 0, 15, 30 and 60 mg/kg bw/day. As no dose relation in incidence and severity was evident, the findings were considered to be unrelated to treatment. The remainder of findings was within the range of normal background lesions which may be recorded in animals of this age and strain.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
Fertility indices were 70, 100, 100 and 90% at 0, 15, 30 and 60 mg/kg bw/day, respectively. The decrease in the control and high dose group was considered to be incidental.
Copulation indices were 100, 90, 90 and 100% at 0, 15, 30 and 60 mg/kg bw/day and delivery indices were 100% for all control and treatment groups.
Gestation length and pre-coital interval: There were no treatment related changes noted for the duration of precoital interval and the duration of gestation.
Number of implantation sites: There were no treatment-related findings.
Number of corpora lutea: There were no treatment-related findings.
Pre-implantation losses: There were no treatment-related findings.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no systemic toxicity observed
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive/fertility
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect observed
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
60 mg/kg bw/day: The pups of one female had cold bodies and an intact umbical cord, indicating lack of nursing behaviour of the female. The finding was limited to a single female and was within the range of historical background data, therefore not attributed to treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Post-implantation losses: There was in increase (statistically not significant) in the percent of post-implantation loss at 15, 30 and 60 mg/kg bw/day (3.31 ± 4.14%, 6.94 ± 11.14%, 14.42 ± 23.93% and 10.56 ± 7.50% at 0, 15, 30 and 60 mg/kg bw/day, respectively). The values were within those of historical control data and without dose relation, therefore the finding was considered to be unrelated to treatment.
Viability indices were 100, 100, 100 and 87.50% at 0, 15, 30 and 60 mg/kg bw/day. The decrease at 60 mg/kg bw/day was within the range of historical control data and thus considered to be incidental.
Number of still births: At 60 mg/kg bw/day a slight increase in the number of stillbirths was noted. The finding was limited to 2 isolated females and within the range of historical control data and therefore considered to be not treatment-related
Number of live pups on PND 0 and 4: A slight decrease in the number of live pups was noted at 60 mg/kg bw/day on PND 4. The finding was attributed to one female of which all pups were found dead between PND 1-2 and not considered treatment-related.
Number of male and female pups on PND 0 and 4: A slight decrease in the total number of male pups was noted at 60 mg/kg bw/day on PND 4. The finding was attributed to one female wherein all pups were found dead between PND 1-2. The observation was within the historical control range and not considered treatment-related.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was a very slight decrease in male litter weight on PND 0 and 4. This was due to a slightly lower number of male pups at 60 mg/kg bw/day, which was compensated by a slightly higher number of female pups.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
0 mg/kg bw/day: one pup of one female had a discoloured dark snout, not treatment-related.
15 mg/kg bw/day: one pup of one female had a discoloured dark snout, not treatment-related.
30 mg/kg bw/day: one pup of one female had a discoloured dark snout, not treatment-related.
60 mg/kg bw/day: one pup of one female had a discoloured dark snout, one pup of one female had a discoloured dark head, one pup of one female had a discoloured dark right hindleg, all pups of one female showed hypothermia and had an intact umbical cord, not treatment-related.
Histopathological findings:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Critical effects observed:
no
Reproductive effects observed:
not specified

Table 1: Clinical signs in parental animals (P0)

Dose group [mg{kg bw/day] 0 15 30 60
Clinical Finding in males
Total number of animals examined 10 10 10 10
Alopecia on flank, thigh and forepaws - 1 - -
Crust on left ear - - - 1
Moderate abnormal breathing - - 1 2
Moderate diarrhoea 7 7 8 7
Moderate piloerection - - 2 4
Moderate salivation - 5 10 10
Moving the bedding - 8 10 10
Nasal discharge (plain/ red) - - 2 4
Severe abnormal breathing - - 1 2
Severe diarrhoea - 1 - -
Severe piloerection - - - 2
Severe salivation - 3 6 8
Slight abnormal breathing - 2 3 2
Slight diarrhoea 7 6 5 7
Slight piloerection - 1 6 4
Slight salivation 2 5 7 10
slightly discoloured faeces (green) - - - 1
Clinical Finding in females
Total number of animals examined 10 10 10 10
Alopecia on head - - - 1
Alopecia on left hind flank - 1 - -
Crust on eye - - 1 -
Moderate abnormal breathing - - - 5
Moderate diarrhoea - 2 5 7
Moderate piloerection 1 - 1 4
Moderate salivation 4 4 8 10
Moderate piloerection - 1 - -
Moving the bedding - 7 10 10
Nasal discharge (red) 2 3 - 2
Severe abnormal breathing - - - 1
Severe diarrhoea - 1 1 2
Severe piloerection - - - 2
Severe salivation - 3 6 8
Slight abnormal breathing 1 - 2 4
Slight diarrhoea - 2 3 -
Slight piloerection - - 6 7
Slight salivation 5 5 6 10
Weight loss - - - 1

Table 2: Body weight development in parental males during treatment

Dose
[mg/kg bw/day]
Males Females
Premating Mating and Postmating Terminal Sacrifice Premating Gestation Lactation
Day 1 Day 7 Day 14 Day 7 Day 14 Day 1 Day 7 Day 14 Day 0 Day 7 Day 14 Day 20 Day 0 Day 4
0 Mean 288 310 328.8 341 352.2 356.7 199.3 201.9 210.7 212.43 237.57 264.57 316.14 244.14 254.29
SD 10.81 10.13 13.69 14.73 19.98 18.77 8.63 9.1 10.97 9.36 9.34 10.13 12.29 21.39 19.36
N 10 10 10 10 10 10 10 10 10 7 7 7 7 7 7
15 Mean 287.9 308.9 326.7 339.3 349.7 352.3 199.5 209.9 209.4 213.67 238.89 270.67 331.11 247.3 256.8
SD 11.47 12.34 15.7 16.42 19.36 19.76 8.86 11.32 7.38 7.98 8.64 11.32 16.24 20.63 15.01
N 10 10 10 10 10 10 10 10 10 9 9 9 9 10 10
% 100 100 99 100 99 99 100.1 104 99.4 100.6 100.6 102.3 104.7 101.3 101
30 Mean 288.8 310.8 330.9 341.7 352.6 355.9 199.8 206 208.7 212 231.5 259.25 310.75 247.67 248.78
SD 12.35 13.45 11.06 13.87 13.5 13.67 8.5 9.03 8.59 9.6 12.11 15.04 27.07 17.8 16.85
N 10 10 10 10 10 10 10 10 10 9 8 8 8 9 9
% 100 100 101 100 98 100 100.3 102 99.1 99.8 97.4 98 98.3 101.4 97.8
60 Mean 290.1 299.3 313.10* 323.90* 327.40* 331.20* 200.8 206.8 217.2 213.33 232.22 261.78 313.89 246.56 248.38
SD 12.01 13.04 15.26 13.76 21.49 20.63 9.1 9.39 12.14 6.44 9.16 10.08 11.67 12.79 8.83
N 10 10 10 10 10 10 10 10 10 9 9 9 9 9 8
% 101 97 95 95 93 93 100.8 102.4 103.1 100.4 97.7 98.9 99.3 101 97.7

Table 3: Food consumption data in parental males and females

Dose
[mg/kg bw/day]
Premating Premating Gestation Lactation
Day 1-7 Day 7-14 Day 1-7 Day 7-14 Day 0-7 Day 7-14 Day 14-20 Day 0-4
0 Mean 155.7 180.9 98.9 116.7 130.57 149.71 140.86 104
SD 12.97 9.76 8.97 12.44 18.89 12.84 15.39 17.39
N 10 10 10 10 7 7 7 7
15 Mean 150.4 176.1 96.7 119 138.56 156.11 144.22 116.6
SD 10.63 10.9 9.6 8.47 45.35 14.16 16.95 18.65
N 10 10 10 10 9 9 9 10
30 Mean 142.2 178.44 102 109.1 134.75 141.88 132 95.56
SD 6.27 7.4 15.3 16.96 24.69 9.03 8.77 25.09
N 10 9 10 10 8 8 8 9
60 Mean 116.20*** 154.50** 96.3 80.29* 169.44 148.33 127.78 96.13
SD 18.47 24.9 7.76 48.26 34 42.18 34.86 32.67
N 10 10 10 7 9 9 9 8

Table 4: Absolute and relative organ weights in parental animals after treatment

Dose
[mg/kg bw/day]
Body weight (g) Males Females
Absolute organ weights Relative organ weights Absolute organ weights Relative organ weights
Prostate# Testes Epididymis Prostate# Ovaries Uterus with Cervix Ovaries Uterus with Cervix
Testes Epididymis left right total left right total
0 Mean 356.7 3.582 1.154 2.097 1.005 0.323 0.588 0.0528 0.05 0.1028 0.941 0.0208 0.0196 0.0404 0.375
SD 18.77 0.398 0.116 0.1932 0.106 0.025 0.051 0.0118 0.012 0.0235 0.12 0.0038 0.0038 0.0075 0.072
N 10 10 10 10 10 10 10 7 7 7 7 7 7 7 7
15 Mean 352.3 3.717 1.203 2.1767 1.056 0.342 0.619 0.058 0.0552 0.1132 0.913 0.0228 0.0216 0.0444 0.357
SD 19.76 0.307 0.072 0.237 0.08 0.02 0.07 0.0059 0.0096 0.0103 0.136 0.003 0.0034 0.0043 0.044
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
% 98.8 103.8 104.2 103.8 105.1 105.7 105.2 109.9 110.3 110.1 97 109.9 109.9 109.9 95.1
30 Mean 355.9 3.615 1.207 2.1044 1.016 0.339 0.592 0.0537 0.0463 0.1 0.94 0.0217 0.0187 0.0403 0.378
SD 13.67 0.253 0.092 0.224 0.064 0.024 0.061 0.0084 0.0051 0.0101 0.115 0.0038 0.0022 0.0047 0.045
N 10 10 10 10 10 10 10 9 9 9 9 9 9 9 9
% 99.8 100.9 104.6 100.4 101.1 104.9 100.6 101.7 92.6 97.3 99.8 104.4 95.1 99.9 100.9
60 Mean 331.2 3.463 1.129 1.8508* 1.046 0.341 0.559 0.0531 0.0529 0.106 0.973 0.0214 0.0213 0.0426 0.392
SD 20.63 0.284 0.108 0.2221 0.063 0.027 0.065 0.0154 0.0103 0.0217 0.122 0.006 0.0036 0.008 0.048
N 10 10 10 10 10 10 10 8 8 8 8 8 8 8 8
% 92.9 96.7 97.9 88.3 104.1 105.5 95.1 100.5 105.9 103.1 103.3 102.8 108.3 105.5 104.5
# = including seminal vesicle and coagulating gland
Conclusions:
There was no systemic toxicity observed. Clinical signs of toxicity, changes in body weight development and food consumption, as well as any pathological findings observed were due to the local irritating effects caused by the corrosive nature of the test item. The NOAEL for both systemic toxicity and fertility of adult animals (P0, male and female) was 60 mg/kg bw/day. The NOAEL for developmental toxicity (F1) was 60 mg/kg bw/day.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable (Klimisch score 1) study performed with the registered substance, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, of Regulation (EC) No. 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A reproduction/developmental toxicity screening test was performed in male and female Wistar rats according to OECD guideline 421 (version adopted in 1995) and in compliance with GLP (2013-0394-DGR). The test item was dissolved in polyethylene glycol (PEG 400) and administered daily by oral gavage to groups of 10 rats per sex. Based on the results of a preliminary dose range-finding study in which mortality was noted at 100 and 125 mg/kg bw/day, dose levels of 15, 30 and 60 mg/kg bw/day were selected for the present study. A similar constituted group of rats received the vehicle and served as control. The animals were treated with the test item or vehicle on a 7 days/week basis, starting 14 days prior to mating. Treatment was continued during the mating period, during gestation and lactation. Males were dosed until the minimum total dosing period of 28 days was completed, while females were dosed up to post-natal day 3. The occurrence of clinical signs and mortality was recorded daily, while body weight and food consumption was recorded weekly. In addition, reproductive and offspring viability parameters were recorded. At scheduled necropsy, selected organs were weighed and all parental animals were subjected to macroscopic and histopathological examinations.

The stability, homogeneity and the concentration of the test item in the vehicle were confirmed by analytical methods. One of ten females at 30 mg/kg bw/day died on gestation day 5 and 1/10 females at 60 mg/kg bw/daywas killed on postnatal day 2 to avoid unnecessary suffering. Both deaths were attributed to the accidental influx or regurgitation of the test item into the respiratory tract and not considered to be toxicologically relevant. Males and females treated with the test item showed an increased incidence and/or severity of moving the bedding, moderate to severe salivation, piloerection and abnormal breathing, compared with the control, which were observed at a higher frequency during the second part of the study (after pre-mating and mating). An increased incidence of nasal discharge was noted in males of the mid- and high-dose groups, compared with the control. In addition, severe diarrhea was noted in 1-2 females per group of the treated groups only. Findings of abnormal breathing, piloerection and nasal discharge were transient although treatment-related. These effects and incidences of moving the bedding and salivation were attributed to the discomfort caused to the animals during the test item treatment. Other findings such as alopecia, eschar and slightly discolored green faeces in individual animals 30 and 60 mg/kg bw/day were considered to be incidental.

A statistically significantly decreased body weight development was noted in males of the high-dose group after 3 weeks of treatment. The findings were consistent with reduced food consumption in males during pre-mating and considered to be treatment-related. However, the observations were considered to be a result of local irritative effects of the test item in the forestomach rather than a sign of systemic toxicity. A statistically significant increase and decrease in body weight gain was noted after the first and second week of treatmentin females at 15 mg/kg bw/day and a statistically significant increase was noted in females at 30 and 60 mg/kg bw/day during the first week of gestation and lactation. The observations on body weight development in females were transient, not dose-related and therefore considered to be without biological relevance. Decreased food consumption in females after two weeks of treatment, during the last week of gestation and during lactation were within the range of normal biological variation.

Gross necropsy and organ weight analysis revealed no treatment-related findings. Although a statistically significant increase in absolute prostate weight was noted in males of the high-dose group, there were no histopathological findings and therefore the observation was not attributed to treatment. At macroscopic examination, reddish discoloured lungs were observed in the female that died during the administration period at 30 mg/kg bw/day and gaseous distention of the caecum and bloody nasal discharge were noted in the female in the high-dose group that was terminated on postnatal day 2. At microscopical examination, both the above-mentioned females exhibited necrotising inflammation in the respiratory tract (trachea, nose and/or bronchi and alveolar areas of the lung) and histopathological findings in the forestomach including epithelial squamous hyperplasia, hyperkeratosis, parakeratosis, submucosal edema and/or dyskeratosis. The findings were attributed to local irritation caused by the test substance. Thymic atrophy and/or splenic lymphoid atrophy and adrenocortical diffuse hypertrophy observed in these females were related to the stress experienced by the animals. In males, sertoli cell vacuolisation and/or spermatid retention were recorded at minimal severity among animals of all groups; however, due to a lacking dose response in incidence and severity the findings were considered to be unrelated to treatment. There was no effect on parental reproductive performance, including copulation, fertility, and delivery indices. The duration of pre-coital intervals and gestation were comparable for all treatment and control groups and the number of corpora lutea and implantation sites was not affected by treatment. In addition, no toxicologically relevant alterations in offspring viability indices were observed.

Based on these results and under the conditions of this study, a NOAEL for toxicity to reproduction (fertility) of 60 mg/kg bw/day was derived for F0 males and females.

Effects on developmental toxicity

Description of key information

Toxicity to reproduction (OECD 421, Reproduction/Developmental Toxicity Screening Test), rat (m, f): NOAEL developmental ≥ 60 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable (Klimisch score 1) study performed with the registered substance, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, of Regulation (EC) No. 1907/2006.
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

An oral gavage reproduction/developmental toxicity screening test was performed according to OECD guideline 421 and in compliance with GLP (2013-0394-DGR). The study has been discussed in detail above under “Toxicity to reproduction”. Here, only findings related to developmental toxicity are discussed.

No treatment-related changes in litter parameters were observed. The total number of pups born, the number of live and stillbirths, the number of male and female pups on postnatal days (PND) 0 and 4 and the sex ratio were not affected by treatment. A slight increase in the percentage of post-implantation loss was noted among the treatment groups, but there was no dose response relationship and all observations remained within the range of historical control data. The slight increase in the number of stillbirths and the slight decrease in the number of live pups and male pups on PND 4 at 60 mg/kg bw/day were attributed to one female wherein all pups were found dead between PND 1-2. The observation was not considered treatment-related. Pup viability indices were 100, 100, 100 and 87.50% at 0, 15, 30 and 60 mg/kg bw/day. The decrease at 60 mg/kg bw/day was within the range of historical control data and thus considered to be incidental.

In addition, there wereno adverse changes of toxicological relevance noted for pup mean weight, total litter weight, male litter weight and female litter weight on PND 0 and 4.A very slight decrease in male litter weight was noted on PND 0 and 4; however, the observation was due a slightly lower number of male pups at 60 mg/kg bw/day, which was compensated by a slightly higher number of female pups.

All the pups of one female at 60 mg/kg bw/day were hypothermic and had an intact umbical cord, indicating lack of nursing behaviour of the female. These pups died on PND 2-3. There was no clear explanation for the occurrence, but the incidence falls within the historical control range. Gross necropsy findings of the offspring revealed a discoloured snout in each one pup at 0, 15, 30 and 60 mg/kg bw/day, and each one pup with a discoloured dark head and a discoloured dark hind leg from one female at 60 mg/kg bw/day. The macroscopical findings were not attributed to treatment.

In conclusion, treatment with the test item did not induce any test item-related changes in litter size, litter viability, pup weight and general condition. No teratogenic effects were noted. Based on the absence of adverse effects, a NOAEL for developmental toxicity was derived to be 60 mg/kg bw/day.

Justification for classification or non-classification

The available data on reproduction and developmental toxicity do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.

No classification for toxicity to reproduction is required according to the criteria of the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations.

Additional information