Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The following information is taken into account for any hazard / risk assessment:

Information from the repeated dose oral study in rats does not suggest any effects on male or female sex organs even at systemic toxic dose levels. In the 28-day repeated dose study organ weights of testes, epididymes and ovaries were comparable to controls and no histopathological chages were observed in these organs up to the highest dose tested. Additional information on fertility is therefore not regarded as priority at this point in time. According to the review Mangelsdorf and Buschmann (2002) who compared information obtained in repeated dose studies on male fertility with those obtained in fertility studies, a 28 -day repeated dose study in which weights of testes and histopathology is determined is already sufficient to detect effects on male fertility. They also conclude that if at the highest dose levels, which produce significant toxicity in other organ systems, no effects on any of these parameters have been found, it can be concluded with a high level of confidence, that a compound is not a male reproductive

toxicant. Further testing on this endpoint should be considered to be of low priority.

 

References:

Mangesldorf I, Buschman J, Federal Institute of Occupational safety and Health, Extrapolation from Results of Animal studies to humans for the endpoint male fertility, Dortmund, Berlin, Dresden 2002).

Aulmann W., Regul Toxicol Pharmacol.2012 Jul;63(2):286-90. Epub 2012 Mar 28.

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
The following information is taken into account for any hazard / risk assessment:
Information from the repeated dose oral study in rats does not suggest any effects on male or female sex organs even at systemic toxic dose levels. In the 28-day repeated dose study organ weights of testes, epididymes and ovaries were comparable to controls and no histopathological chages were observed in these organs up to the highest dose tested. Additional information on fertility is therefore not regarded as priority at this point in time. According to the review Mangelsdorf and Buschmann (2002) who compared information obtained in repeated dose studies on male fertility with those obtained in fertility studies, a 28 -day repeated dose study in which weights of testes and histopathology is determined is already sufficient to detect effects on male fertility. They also conclude that if at the highest dose levels, which produce significant toxicity in other organ systems, no effects on any of these parameters have been found, it can be concluded with a high level of confidence, that a compound is not a male reproductive
toxicant. Further testing on this endpoint should be considered to be of low priority.

References:
Mangesldorf I, Buschman J, Federal Institute of Occupational safety and Health, Extrapolation from Results of Animal studies to humans for the endpoint male fertility, Dortmund, Berlin, Dresden 2002).
Aulmann W., Regul Toxicol Pharmacol.2012 Jul;63(2):286-90. Epub 2012 Mar 28.
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The information from the 28-day study is considered screening level information on fertility that is considered as posing no immediate concern for effects on fertility.
Additional information

Information from the repeated dose oral study in rats does not suggest any effects on male or female sex organs even at systemic toxic dose levels. In the 28-day repeated dose study organ weights of testes, epididymes and ovaries were comparable to controls and no histopathological chages were observed in these organs up to the highest dose tested. Additional information on fertility is therefore not regarded as priority at this point in time. According to the review Mangelsdorf and Buschmann (2002) who compared information obtained in repeated dose studies on male fertility with those obtained in fertility studies, a 28 -day repeated dose study in which weights of testes and histopathology is determined is already sufficient to detect effects on male fertility. They also conclude that if at the highest dose levels, which produce significant toxicity in other organ systems, no effects on any of these parameters have been found, it can be concluded with a high level of confidence, that a compound is not a male reproductive

toxicant. Further testing on this endpoint should be considered to be of low priority.

References:

Mangesldorf I, Buschman J, Federal Institute of Occupational safety and Health, Extrapolation from Results of Animal studies to humans for the endpoint male fertility, Dortmund, Berlin, Dresden 2002).

Aulmann W., Regul Toxicol Pharmacol.2012 Jul;63(2):286-90. Epub 2012 Mar 28.



Short description of key information:
Screening information from the repeated dose study does not suggest any effects on male or female sex organs even at systemic toxic dose levels. In the 28-day repeated dose study organ weights of testes, epididymes and ovaries were comparable to controls and no histopathological chages were observed in these organs up to the highest dose tested. Additional information on fertility is therefore not regarded as priority at this point in time.

Justification for selection of Effect on fertility via oral route:
No effects on the reproductive organs of male and female animals were observed at systemic toxic dose levels. Organ weights and histopathology of the reproductive organs were not affected.

Effects on developmental toxicity

Description of key information

Within the context of the developmental toxicity study, the No Observed Effect Level (NOEL) for the pregnant females was 3 mg/kg bw/day A.I. It was not possible to identify a NOEL for the foetus due to treatment related changes observed for the foetal kidneys and ureters at a dosage as low as 3 mg/kg bw/day A.I. which appear to represent a selective effect of treatment on the developing conceptus.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 February 2013 to 15 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
January 22, 2001
Deviations:
yes
Remarks:
Deviations the 5 hour observation period could not be performed and was performed earlier at one occasion. Food consumptuionm data for day 5 were lost for 22 animals. Food consuption was calculated between day 6 and 8.
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147 (24 November 2000)
Principles of method if other than guideline:
As food consumption data is calculated as an average consumption over a period of days, thecalculations were adjusted accordingly and the loss of data for Day 5 considered not to affect the purpose or integrity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
Identification: SD10
Description: Clear colourless liquid
Chemical Name: N,N-di-(3,3-dimethyl-2-butyl)-1,6-diaminohexane
Chemical Formula: C18H40N2
Batch: 9147-192-3a
Purity: 96.9% +
Date Received: 16 January 2012
Expiry Date: 01 May 2013
Storage Conditions: Room temperature in the dark, under nitrogen
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd., Margate, Kent
- Time mated females at day 0 or day 1 of gestation
- Weight at study initiation: 195 to 297 g
- Fasting period before study:
- Housing: single air conditioned room, individual solid floor polypropylene cages with softwood flakes and stailess steel lids.
- Diet ad libitum
- Water ad libitum
- Acclimation period: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3 deg. C
- Humidity (%): 50 +- 20
- Air changes (per hr): at least 15 air changes per h
- Photoperiod (hrs dark / hrs light): 12 h dark/ 12 h light

IN-LIFE DATES: From: To:19.02. 2013 to 05.03.2013
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): the vehicle was found suitable in other previous studies
- Concentration in vehicle: 0.75, 1.5, 3 mg/mL
- Amount of vehicle (if gavage): 4 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken of each test item formulation and were analysed for the test substance concentration at Harlan analytical Laboratrory, Sharlow. Stability and homogeneity of the test item solutions were previously determined by the same laboratory (project No. 1193/0014) and were found to be stable for at least 14 days. Prepared formulations were within 95 to 108% of the nominal concentrations.
Details on mating procedure:
96 time mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River Laboratories at day 0 or day 1 of gestation. For each animal the day that positive evidence of mating was observed at the animal supplier was designated Day 0 of gestation. On arrival the females weighed 195 to 297g.
Duration of treatment / exposure:
The test item was administered daily, from Day 5 to Day 19 of gestation, by gavage. Control animals were treated in an identical manner with the vehicle alone.
Frequency of treatment:
Daily
Duration of test:
The in-life phase of the study was conducted between 15 February 2013 (first day of treatment) and 07 March 2013 (final necropsy).
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
3 mg/kg bw/day (nominal)
Remarks:
Low dose group
Dose / conc.:
6 mg/kg bw/day (nominal)
Remarks:
Intermediate dose group
Dose / conc.:
12 mg/kg bw/day (nominal)
Remarks:
High dose group
No. of animals per sex per dose:
24 females per dose group = 96 female rate in total
Control animals:
yes, concurrent vehicle
Details on study design:
The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels of 3, 6, and 12 mg/kg bw/day Active Ingredient (A.I.) (incorporating a correction factor for 97.17% purity). A further group of twenty-four time mated females was exposed to the vehicle only (Arachis Oil BP) over the same treatment period to serve as a control.
Clinical signs, body weight change, food and water consumptions were monitored during the study. Liver, kidney and adrenal weights were recorded for all females at termination and histopathological evaluation of the liver, kidneys and adrenals was performed.
All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, foetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were preserved in Industrial Methylated Spirit (IMS) and then transferred into 100% glycerol and examined for skeletal development. The remaining half were preserved in Bouin’s solution and examined viscerally.
Maternal examinations:
Mortality: There were no unscheduled deaths during the study.
Clinical Observations: Clinical signs did not indicate any effect of treatment at 3, 6 and 12 mg/kg bw/day A.I.
Body Weight: At 12 mg/kg bw/day A.I. cumulative body weight gain was lower than control from Day 11 of gestation, and overall body weight gain even after adjustment for the gravid uterus, remained significantly lower than control.
Body weight gain of pregnant females at 3 and 6 mg/kg bw/day A.I. was considered to have been unaffected by treatment.
Food Consumption: At 12 mg/kg bw/day A.I. food consumption was lower than control from Day 11 of gestation. At 3 and 6 mg/kg bw/day A.I. food intake was considered to have been unaffected by treatment.
Post Mortem Studies: Necropsy examination on Day 20 of gestation revealed enlarged adrenals for all females receiving 6 and 12 mg/kg bw/day A.I.
Organ Weight: At 6 and 12 mg/kg bw/day A.I. absolute and body weight relative adrenals were statistically significantly higher than control.
Histopathology: The following treatment-related microscopic abnormalities were detected:
Liver: Single cell necrosis and focal necrosis was evident in females treated with 12 mg/kg bw/day A.I. Although single cell necrosis was also present in occasional females treated with 3 or 6 mg/kg bw/day A.I., this was at minimal severity (often only affecting a single cell) and was considered to be an incidental finding.
Adrenals: Hypertrophy, lymphocytic infiltrates and single cell necrosis was evident in the adrenal cortex of females treated with 12 and 6 mg/kg bw/day A.I. Hemorrhage and sinusoidal dilation was also evident in the adrenal cortex of females treated with 12 mg/kg bw/day A.I.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods
outlined below:
Body weight and body weight change (including adjustment for the contribution of the gravid uterus), food consumption, gravid uterus weight, litter data and foetal litter and placental weights: Bartlett’s test for homogeneity of variance. Where the data were shown to be homogeneous one way analysis of variance and, if significant, Dunnett’s multiple comparison test or, was employed, where the data were found to non homogeneous Kruskal-Wallis and, if significant, pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test was employed.
Foetal evaluation parameters, including skeletal or visceral findings were analysed by Kruskal-Wallis and, if significant, Mann-Whitney ‘U’ test.
Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Indices:
All data was summarised in tabular form, including reproductive incides. As the litter is the standard unit of assessment mean values and incidences were first calculated within each litter and then groups mean values calculated from these litter values. Group mean values are calculated from unrounded values, values in appendices may represent rounded values for presentation purposes therefore it may not always be possible to calculate the exact group mean values from those presented within the appendices.

Pre and Post Implantation Loss calculations are as follows:
Percentage pre-implantation loss was calculated as: (number of corpora lutea - number of implantations÷ number of corpora lutea) x 100
Percentage post-implantation loss was calculated as: (number of implantations - number of live foetuses÷ number of implantations) x 100
Sex ratio was calculated as: % male foetuses (sex ratio) = (Number of male foetuses ÷ Total number of foetuses) x 100

Clinical signs:
no effects observed
Description (incidence and severity):
The low number of clinical signs observed for adult animals on the study did not indicate any
obvious adverse effect of treatment at 3, 6 and 12 mg/kg bw/day.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At 12 mg/kg bw/day A.I. cumulative body weight gain during gestation tended to be lower than control from Day 11 of gestation, with differences frequently attaining statistical significance. Although this may in part reflect lower foetal/litter weight at this dosage, final body weight and overall body weight gain after adjustment for the gravid uterus remained significantly lower than control.
At 3 and 6 mg/kg bw/day A.I. there was no adverse effect of treatment on body weight gain of the pregnant females. At 6 mg/kg bw/day A.I. slightly lower body weight gain between Days 8 and 11 attained statistical significance when compared to control but, in isolation, and in the absence of any effect on cumulative body weight gain this finding was considered most likely to reflect normal biological variation.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
At 12 mg/kg bw/day A.I. food consumption during gestation was lower than control from Day 11 of gestation, with differences frequently attaining statistical significance. This lower food intake was consistent with the lower body weight gain observed for these animals at this time.
At 3 and 6 mg/kg bw/day A.I. there were no consistent differences from control for food intake that indicated an adverse effect of treatment.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Immunological findings:
not examined
Neuropathological findings:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Effects on pregnancy duration:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Key result
Dose descriptor:
LOAEL
Effect level:
6 mg/kg bw/day (nominal)
Based on:
act. ingr.
Basis for effect level:
other: increased relative adernal weights and adenal histopathological changes (lymphocytic infiltrates, single cell necrosis, hypotrophy)
Remarks on result:
other:
Remarks:
see basis for effect level
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 12 mg/kg bw/day A.I. maternal treatment was associated with lower mean foetal and litter weights and increased mean placental weights compared to control.
At 6 mg/kg bw/day A.I. maternal treatment was associated with lower mean foetal weights and to a lesser extent, litter weights, compared to control.
At 3 mg/kg bw/day A.I. foetal weights were lower than control.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of implantations, early and late embryonic/foetal deaths, number of male and female foetuses, total number of foetuses or sex ratio were unaffected by maternal treatment at 3, 6 and 12 mg/kg bw/day A.I.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The number of implantations, early and late embryonic/foetal deaths, number of male and female foetuses, total number of foetuses or sex ratio were unaffected by maternal treatment at 3, 6 and 12 mg/kg bw/day A.I.
Fetal/pup body weight changes:
effects observed, treatment-related
Changes in litter size and weights:
effects observed, treatment-related
External malformations:
effects observed, treatment-related
Skeletal malformations:
effects observed, treatment-related
Visceral malformations:
effects observed, treatment-related
Key result
Dose descriptor:
LOAEL
Effect level:
ca. 3 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
visceral malformations
Remarks on result:
not determinable
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
3 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

There was no obvious adverse effect of maternal treatment on litter data as assessed by the number of implantations, early and late embryonic/foetal deaths, number of male and female foetuses, total number of foetuses or sex ratio at 3, 6 and 12 mg/kg bw/day A.I. At 12 mg/kg bw/day A.I. maternal treatment was associated with lower mean foetal weights and increased mean placental weights compared to control, with differences attaining statistical significance. Mean litter weight was also lower than control, although differences from control did not attain statistical significance. At 6 mg/kg bw/day A.I. maternal treatment was associated with lower mean foetal weights compared to control, with differences attaining statistical significance. Mean litter weight was also lower than control, but differences did not attain statistical significance. Placental weights were not obviously influenced by maternal treatment. At 3 mg/kg bw/day A.I. maternal treatment was associated with lower mean foetal weights compared to control, although no statistically significant differences were apparent for mean female weight or litter weights at this dosage. Placental weights were not obviously influenced by maternal treatment. At 12 mg/kg bw/day A.I. a high incidence of foetuses was found to have generalised subcutaneous oedema at external necropsy examination and this was confirmed during the subsequent more detailed visceral examinations of the foetuses. These visceral examinations also revealed small/no development of renal papilla(e) and kinked and/or dilated ureter(s) for the majority of foetuses (all litters affected) at this dosage. There was also a slight increase in the incidence of foetuses with increased renal pelvic cavitation, with the majority of litters being affected. For male offspring there was also a higher incidence of undescended testes. Detailed skeletal examination at 12 mg/kg bw/day A.I. revealed a plethora of changes to skeletal development compared to that observed for the control. These included an increased incidence of foetus (litters) with large fontanelle, incomplete ossification of cranial bones and facial bone(s), no ossification of hyoid, reduced number of fully ossified sternebrae, post lumber vertebral centra and arches, increased incidence of semi-bipartite thoracic centre, no ossification of one or more sternebra, increased incidence of wavy ribs and short 13th rib, increased incidence of incomplete/no ossification of pubes and reduced number of metacarpals and metatarsals. At 6 mg/kg bw/day A.I. only one foetus showed generalised subcutaneous oedema and this finding was not confirmed during more detailed examination of the offspring. However, detailed visceral evaluations did reveal small/no development of renal papilla(e) and kinked and/or dilated ureter(s) for the majority of foetuses (all litters affected), similar to that seen in high dosage foetuses. Again, there was also a slight increase in the incidence of foetuses (5 litters affected) with increased renal pelvic cavitation, although the incidence was lower than had been observed at 12 mg/kg bw/day A.I. Detailed skeletal examination at 6 mg/kg bw/day A.I. also revealed a number of changes to skeletal development compared to that observed for the control, although the incidence and number of findings was lower than observed at 12 mg/kg bw/day A.I. These findings included an increased incidence of foetus (litters) with large fontanelle, incomplete ossification of facial bone(s), no ossification of hyoid, reduced number of fully ossified sternebrae and post lumber vertebral arches, increased incidence of semi-bipartite thoracic centre, slight increased incidence of wavy ribs and short 13th rib and reduced number of metacarpals and metatarsals.

At 3 mg/kg bw/day A.I. external necropsy examination did not reveal any obvious effect of maternal treatment. Foetuses from a single litter showed encephalocoele, spina bifida and cleft palate but, in isolation, and in the absence of similar finding at higher dosages, this was considered to be genetic in origin and unrelated to maternal treatment. However detailed visceral examinations did reveal an increased incidence of foetuses/litters showing small/no development of renal papilla(e) and kinked and/or dilated ureter(s) similar to that seen at higher dosages. The incidence of findings at detailed skeletal examination at 3 mg/kg bw/day A.I. was generally similar to control. These was a marginal increase in the incidence of foetuses with incomplete ossification of facial bones and reduced number of metacarpals, but these findings did not indicate any significant disturbance of foetal development.

Conclusions:
Within the context of this study, the No Observed Effect Level (NOEL) for the pregnant females was 3 mg/kg bw/day A.I. It was not possible to identify a NOEL for the foetus due to treatment related changes observed for the foetal kidneys and ureters at a dosage as low as 3 mg/kg bw/day A.I. which appear to represent a selective effect of treatment on the developing conceptus.
Executive summary:

A GLP compliant OECD Guideline 414 “Prenatal Developmental ToxicityStudy” (adopted 22 January 2001) was performed to investigate the effects of the test item on embryonic and foetal development following repeated administration by gavage to the pregnant female during the period of organogenesis. The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels of 3, 6, and 12 mg/kg bw/day Active Ingredient (A.I.) (incorporating a correction factor for 97.17% purity). A further group of twenty-four time mated females was exposed to the vehicle only (Arachis Oil BP) over the same treatment period to serve as a control. Clinical signs, body weight change, food and water consumptions were monitored during the study. Liver, kidney and adrenal weights were recorded for all females at termination and histopathological evaluation of the liver, kidneys and adrenals was performed. All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, foetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were preserved in Industrial Methylated Spirit (IMS) and then transferred into 100% glycerol and examined for skeletal development. The remaining half were preserved in Bouin’s solution and examined viscerally.

Maternal responses observed were as follows:There were no unscheduled deaths during the study. Clinical signs did not indicate any effect of treatment at 3, 6 and 12 mg/kg bw/day A.I. At 12 mg/kg bw/day A.I. cumulative body weight gain was lower than control from Day 11 of gestation, and overall body weight gain even after adjustment for the gravid uterus, remained

significantly lower than control. Body weight gain of pregnant females at 3 and 6 mg/kg bw/day A.I. was considered to have been unaffected by treatment. At 12 mg/kg bw/day A.I. food consumption was lower than control from Day 11 of gestation. At 3 and 6 mg/kg bw/day A.I. food intake was considered to have been unaffected by treatment. Necropsy examination on Day 20 of gestation revealed enlarged adrenals for all females receiving 6 and 12 mg/kg bw/day A.I. At 6 and 12 mg/kg bw/day A.I. absolute and body weight relative adrenals were statistically

significantly higher than control. The following treatment-related microscopic abnormalities were detected:

Liver: Single cell necrosis and focal necrosis was evident in females treated with 12 mg/kg bw/day A.I. Although single cell necrosis was also present in occasional females treated with 3 or 6 mg/kg bw/day A.I., this was at minimal severity (often only affecting a single cell) and was considered to be an incidental finding.

Adrenals: Hypertrophy, lymphocytic infiltrates and single cell necrosis was evident in the adrenal cortex of females treated with 12 and 6 mg/kg bw/day A.I. Hemorrhage and sinusoidal

dilation was also evident in the adrenal cortex of females treated with 12 mg/kg bw/day A.I.

Litter responses were noted as follows:

The number of implantations, early and late embryonic/foetal deaths, number of male and female foetuses, total number of foetuses or sex ratio were unaffected by maternal treatment at 3, 6 and 12 mg/kg bw/day A.I. At 12 mg/kg bw/day A.I. maternal treatment was associated with lower mean foetal and litter weights and increased mean placental weights compared to control. At 6 mg/kg bw/day A.I. maternal treatment was associated with lower mean foetal weights and to a lesser extent, litter weights, compared to control. At 3 mg/kg bw/day A.I. foetal weights were lower than control.

Maternal treatment at 12 mg/kg bw/day A.I. was associated with generalised subcutaneous oedema, small/no development of renal papilla(e) and kinked and/or dilated ureter(s) and an

increased incidence of foetuses with increased renal pelvic cavitation. For male offspring there was also a higher incidence of undescended testes. Skeletal examination revealed an increased incidence of foetus (litters) with large fontanelle, incomplete ossification of cranial bones and facial bone(s), no ossification of hyoid, reduced number of fully ossified sternebrae, post lumber vertebral centra and arches, increased incidence of semi-bipartite thoracic centre, no ossification of one or more sternebra, increase incidence of wavy ribs and short 13th rib, increase incidence of incomplete/no ossification of pubes and reduced number of metacarpals and metatarsals.

Maternal treatment at 6 mg/kg bw/day A.I. was associated with small/no development of renal papilla(e) and kinked and/or dilated ureter(s) and a slight increase in the incidence of increased

renal pelvic cavitation. Skeletal examination revealed an increase incidence of foetus (litters) with large fontanelle, incomplete ossification of facial bone(s), no ossification of hyoid, reduced

number of fully ossified sternebrae and post lumber vertebral arches, increased incidence of semi-bipartite thoracic centre, slight increase incidence of wavy ribs and short 13th rib and

reduced number of metacarpals and metatarsals. Maternal treatment at 3 mg/kg bw/day A.I. was associated with an increased incidence of foetuses/litters showing small/no development of renal papilla(e) and kinked and/or dilated ureter(s) similar to that seen at higher dosages. Skeletal examination revealed a marginal increase in the incidence of foetuses with incomplete ossification of facial bones and reduced number of metacarpals, but these findings did not indicate any significant disturbance of skeletal development.

Therefore based on the results of this study the No Observed Effect Level (NOEL) for the pregnant females was 3 mg/kg bw/day A.I. It was not possible to identify a NOEL for the foetus due to treatment related changes observed for the foetal kidneys and ureters at a dosage as low as 3 mg/kg bw/day A.I. which appear to represent a selective effect of treatment on the developing conceptus.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
3 mg/kg bw/day
Species:
rat
Additional information

Justification for selection of Effect on developmental toxicity: via oral route:
For other regulatory purposes a developmental toxicity study was conducted and is available.

Justification for classification or non-classification

Based on the results of the OECD 414 developmental toxicity study conducted in pregnant Sprague-Dawley rats administered 3,6 and 12mg/kg bw/day active ingredient between days 5 and 19 revealed developmental toxicity effects at a dose from 3 mg/kg. The developmental toxicity effects included reduced fetal and litter weights and specific malformations in the kidneys and ureter as well as an increase in skeletal malformations and variations. Effects on the dams were observed from 6 mg/kg bw. Therefore according to CLP Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixture this substance meets the classification criteria for Repro Cat 1B.